U.S. patent application number 11/522253 was filed with the patent office on 2010-10-21 for highly conserved genes and their use to generate probes and primers for detection of microorganisms.
Invention is credited to Michel G. Bergeron, Maurice Boissinot, Ann Huletsky, Christian Menard, Marc Ouellette, Francois Picard, Paul H. Roy.
Application Number | 20100267012 11/522253 |
Document ID | / |
Family ID | 42984085 |
Filed Date | 2010-10-21 |
United States Patent
Application |
20100267012 |
Kind Code |
A1 |
Bergeron; Michel G. ; et
al. |
October 21, 2010 |
Highly conserved genes and their use to generate probes and primers
for detection of microorganisms
Abstract
Compositions and methods for the detection of
vancomycin-resistant pathogens using primers and/or probes to the
vanA and vanB genes.
Inventors: |
Bergeron; Michel G.;
(Quebec, CA) ; Boissinot; Maurice;
(Saint-Augustin-de-Desmaures, CA) ; Huletsky; Ann;
(Sillery, CA) ; Menard; Christian;
(St-Lambert-de-Levis, CA) ; Ouellette; Marc;
(Sillery, CA) ; Picard; Francois; (Cap-Rouge,
CA) ; Roy; Paul H.; (Loretteville, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
42984085 |
Appl. No.: |
11/522253 |
Filed: |
September 14, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11236785 |
Sep 27, 2005 |
|
|
|
11522253 |
|
|
|
|
10089177 |
Mar 27, 2002 |
|
|
|
PCT/CA00/01150 |
Sep 28, 2000 |
|
|
|
11236785 |
|
|
|
|
10753169 |
Jan 7, 2004 |
|
|
|
10089177 |
|
|
|
|
09989643 |
Nov 20, 2001 |
|
|
|
10753169 |
|
|
|
|
09297539 |
May 3, 1999 |
|
|
|
PCT/CA97/00829 |
Nov 4, 1997 |
|
|
|
09989643 |
|
|
|
|
Current U.S.
Class: |
435/6.1 ;
435/6.18; 536/24.33 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/689 20130101; C12Q 2600/16 20130101; G01N 2333/195 20130101;
C12Q 1/14 20130101; G01N 2333/31 20130101; C12Q 2600/166
20130101 |
Class at
Publication: |
435/6 ;
536/24.33 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/00 20060101 C07H021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 28, 1999 |
CA |
2283458 |
May 19, 2000 |
CA |
2037010 |
Claims
1. A composition for the detection of a vancomycin resistant
pathogen in a sample using a nucleic acid amplification assay,
wherein said composition comprises a primer pair for the
amplification of vanA sequences, said primer pair comprising a
forward and a reverse oligonucleotides, wherein said forward and
reverse oligonucleotides are between 13 and 30 nucleotides, and
wherein said forward and reverse oligonucleotides each comprises a
binding region that is complementary to primer binding sites
present on opposite strands of the pathogen's DNA, wherein said
primer pair is adapted to amplify a target region of a vancomycin
resistance gene of said pathogen's DNA between and including said
primer binding sites to produce a detectable vanA vancomycin
amplification product, wherein at least one of said
oligonucleotides comprises, or is fully complementary to, at least
11 consecutive nucleotides of SEQ ID NO: 1090 and SEQ ID NO: 1091,
or the complements thereof.
2. (canceled)
3. (canceled)
4. The composition of claim 1, further comprising at least one
internal hybridization probe, wherein said internal hybridization
probe can hybridize under stringent conditions to said vanA
vancomycin amplification product.
5. The composition of claim 4, wherein said internal hybridization
probe is a molecular beacon.
6. The composition of claim 4, wherein said internal hybridization
probe comprises at least 10 consecutive nucleotides of SEQ ID NO:
2299.
7. (canceled)
8. The composition of claim 4, further comprising: an internal
control DNA; and an internal control probe, wherein said internal
control DNA is amplified using the said forward and reverse
oligonucleotides to produce a control amplification product in said
nucleic acid amplification assay, and wherein said internal control
probe hybridizes to said internal control DNA under the same
conditions as said internal hybridization probe hybridizes to said
vancomycin amplification product.
9. The composition of claim 8, wherein said internal control probe
comprises at least 10 consecutive nucleotides of the sequence of
SEQ ID NO: 2301.
10. A kit comprising the composition of claim 1.
11. A method to detect the presence of vancomycin-resistant
organisms in a sample comprising nucleic acids, comprising:
annealing the nucleic acids of said sample with the composition of
claim 4; amplifying said sample nucleic acids with said annealed
primer pair; and determining the amount of internal hybridization
probe that is annealed to the vanA vancomycin amplification product
and the target region.
12. The method of claim 11, wherein said primer pair consists of
SEQ ID NO's 1090 and 1091, or the complements thereof.
13. The method of claim 11, wherein said primer pair and said
internal hybridization probe are placed in the same physical
enclosure.
14. (canceled)
15. The method of claim 11 wherein amplification step comprises a
method selected from the group consisting of: (a) polymerase chain
reaction (PCR), (b) ligase chain reaction, (c) nucleic acid
sequence-based amplification, (d) self-sustained sequence
replication, (e) strand displacement amplification, (f) branched
DNA signal amplification, (g) nested PCR, and (h) multiplex
PCR.
16. The method of claim 15, wherein said amplification step
comprises PCR.
17. (canceled)
18. The method of claim 11, wherein said internal hybridization
probe comprises at least 10 consecutive nucleotides of the sequence
of SEQ ID NO: 2299.
19. The composition of claim 1, wherein said forward or said
reverse oligonucleotide consists of SEQ ID NO: 1090.
20. The composition of claim 1, wherein said forward or said
reverse oligonucleotide consists of SEQ ID NO: 1091.
21. The composition of claim 1, wherein said forward and reverse
oligonucleotides consist of SEQ ID NO: 1090 and SEQ ID NO: 1091,
respectively.
22. The composition of claim 21, further comprising an internal
hybridization probe, wherein said internal hybridization probe can
hybridize under stringent conditions to said vancomycin
amplification product.
23. The composition of claim 22, wherein said internal
hybridization probe is a molecular beacon.
24. The composition of claim 22, wherein said internal
hybridization probe comprises at least 10 consecutive nucleotides
of the sequence of SEQ ID NO: 2299.
25. The composition of claim 22, further comprising: an internal
control DNA; and an internal control probe, wherein said internal
control DNA is amplified under the same conditions and using said
forward and reverse primers to produce a control amplification
product, and wherein said internal control probe hybridizes to said
internal control DNA under the same conditions as said internal
hybridization probe hybridizes to said vanA vancomycin
amplification product.
26. The composition of claim 25, wherein said internal control DNA
is SEQ ID NO: 2302.
27. The composition of claim 25, wherein said internal control
probe comprises at least 10 consecutive nucleotides of the sequence
of SEQ ID NO: 2301.
28. The composition of claim 8, wherein said internal control DNA
is SEQ ID NO: 2302.
29. The composition of claim 1, further comprising a second
amplification primer pair for the amplification of vanB sequences,
said second primer pair comprising a second forward and a second
reverse oligonucleotide, wherein said second forward and second
reverse oligonucleotides are each between 13 and 30 nucleotides and
wherein each of said forward and reverse oligonucleotides comprises
a binding region that is complementary to primer binding sites
present on opposite strands of the pathogen's DNA, wherein said
primer pair is adapted to amplify a vancomycin resistance gene of
said pathogen's DNA between and including said primer binding sites
to produce a detectable vanB vancomycin amplification product,
wherein at least one of said oligonucleotides of said second primer
pair is selected from the group consisting of: SEQ ID NO: 1095 and
SEQ ID NO: 1096, or the complements thereof.
30. The composition of claim 29, wherein said second forward and
second reverse oligonucleotides of said second primer pair consist
of SEQ ID NO: 1095 and SEQ ID NO: 1096, respectively.
31. The composition of claim 29 further comprising an internal
hybridization probe, wherein said internal hybridization probe can
hybridize under stringent conditions to said vanA vancomycin
amplification product or said vanB vancomycin amplification
product.
32. The composition of claim 31, wherein said internal
hybridization probe is a molecular beacon.
33. The composition of claim 31, wherein said internal
hybridization probe comprises at least 10 consecutive nucleotides
of the sequence of SEQ ID NO: 2299.
34. The composition of claim 31, wherein said internal
hybridization probe comprises at least 10 consecutive nucleotides
of the sequence of SEQ ID NO: 2300.
35. The composition of claim 29, further comprising: an internal
control DNA; and an internal control probe, wherein said internal
control DNA is amplified under the same conditions and using the
same forward and reverse oligonucleotides to produce a control
amplification product, and wherein said internal control probe
hybridizes to said internal control DNA under the same conditions
as said internal hybridization probe hybridizes to said vanA or
vanB vancomycin amplification product.
36. The composition of claim 36, wherein said internal control DNA
comprises SEQ ID NO: 2302.
37. The composition of claim 36, wherein said internal control
probe comprises at least 10 consecutive nucleotides of the sequence
of SEQ ID NO: 2301.
38. The method of claim 11, further comprising annealing the sample
nucleic acids with: an internal control DNA; and an internal
control probe, wherein said internal control DNA is amplified under
the same conditions and using the same forward and reverse
oligonucleotides as to produce a control amplification product, and
wherein said internal control probe hybridizes to said internal
control DNA under the same conditions as said internal
hybridization probe hybridizes to said vanA vancomycin
amplification product.
39. The method of claim 38, wherein said internal control probe
comprises at least 10 consecutive nucleotides of the sequence of
SEQ ID NO: 2301.
40. The method of claim 38, wherein said internal control DNA
comprises of SEQ ID NO: 2302.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 11/236,785, filed Sep. 27, 2005, which is a continuation
of Ser. No. 10/089,177, filed Mar. 27, 2002, which is the U.S.
national phase under 35 U.S.C. .sctn.371 of prior PCT International
Application No. PCT/CA00/01150, filed Sep. 28, 2000, which claims
the benefit of Canada Application No. 2037010 filed May 19, 2000,
and Canada Application No. 2283458, filed Sep. 28, 1999, the
disclosures of which are hereby expressly incorporated by reference
in their entireties. This application is also a
continuation-in-part of application Ser. No. 10/753,169, filed Jan.
7, 2004, which is a continuation of application Ser. No.
09/989,643, filed Nov. 20, 2001 which is a continuation of
application Ser. No. 09/297,539 filed May 3, 1999 which is a
national phase of PCT/CA97/00829 filed Nov. 4, 1997, which claims
priority to application Ser. No. 08/743,637, filed Nov. 4, 1996,
the disclosures of which are hereby expressly incorporated by
reference in their entireties.
SEQUENCE LISTING
[0002] The present application is being filed along with duplicate
copies of a CD-ROM marked "Copy 1" and "Copy 2" containing a
Sequence Listing in electronic format. The duplicate copies of the
CD-ROM each contain a file entitled GENOM.048NPCCP1.TXT created on
Sep. 14, 2006 and is 1,929,380 bytes in size. The information on
these duplicate CD-ROMs is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
Classical Methods for the Identification of Microorganisms
[0003] Microorganisms are classically identified by their ability
to utilize different substrates as a source of carbon and nitrogen
through the use of biochemical tests such as the API20E.TM. system
(bioMerieux). For susceptibility testing, clinical microbiology
laboratories use methods including disk diffusion, agar dilution
and broth microdilution. Although identifications based on
biochemical tests and antibacterial susceptibility tests are
cost-effective, generally two days are required to obtain
preliminary results due to the necessity of two successive
overnight incubations to identify the bacteria from clinical
specimens as well as to determine their susceptibility to
antimicrobial agents. There are some commercially available
automated systems (i.e. the MicroScan.TM. system from Dade Behring
and the Vitek.TM. system from bioMerieux) which use sophisticated
and expensive apparatus for faster microbial identification and
susceptibility testing (Stager and Davis, 1992, Clin. Microbiol.
Rev. 5:302-327). These systems require shorter incubation periods,
thereby allowing most bacterial identifications and susceptibility
testing to be performed in less than 6 hours. Nevertheless, these
faster systems always require the primary isolation of the bacteria
or fungi as a pure culture, a process which takes at least 18 hours
for a pure culture or 2 days for a mixed culture. So, the shortest
time from sample reception to identification of the pathogen is
around 24 hours. Moreover, fungi other than yeasts are often
difficult or very slow to grow from clinical specimens.
Identification must rely on labor-intensive techniques such as
direct microscopic examination of the specimens and by direct
and/or indirect immunological assays. Cultivation of most parasites
is impractical in the clinical laboratory. Hence, microscopic
examination of the specimen, a few immunological tests and clinical
symptoms are often the only methods used for an identification that
frequently remains presumptive.
[0004] The fastest bacterial identification system, the
autoSCAN-Walk-Away.TM. system (Dade Behring) identifies both
gram-negative and gram-positive bacterial species from standardized
inoculum in as little as 2 hours and gives susceptibility patterns
to most antibiotics in 5 to 6 hours. However, this system has a
particularly high percentage (i.e. 3.3 to 40.5%) of non-conclusive
identifications with bacterial species other than
Enterobacteriaceae (Croize J., 1995, Lett. Infectiol. 10:109-113;
York et al., 1992, J. Clin. Microbiol. 30:2903-2910). For
Enterobacteriaceae, the percentage of non-conclusive
identifications was 2.7 to 11.4%. The list of microorganisms
identified by commercial systems based on classical identification
methods is given in Table 15.
[0005] A wide variety of bacteria and fungi are routinely isolated
and identified from clinical specimens in microbiology
laboratories. Tables 1 and 2 give the incidence for the most
commonly isolated bacterial and fungal pathogens from various types
of clinical specimens. These pathogens are the main organisms
associated with nosocomial and community-acquired human infections
and are therefore considered the most clinically important.
Clinical Specimens Tested in Clinical Microbiology Laboratories
[0006] Most clinical specimens received in clinical microbiology
laboratories are urine and blood samples. At the microbiology
laboratory of the Centre Hospitalier de l'Universite Laval (CHUL),
urine and blood account for approximately 55% and 30% of the
specimens received, respectively (Table 3). The remaining 15% of
clinical specimens comprise various biological fluids including
sputum, pus, cerebrospinal fluid, synovial fluid, and others (Table
3). Infections of the urinary tract, the respiratory tract and the
bloodstream are usually of bacterial etiology and require
antimicrobial therapy. In fact, all clinical samples received in
the clinical microbiology laboratory are tested routinely for the
identification of bacteria and antibiotic susceptibility.
Conventional Pathogen Identification from Clinical Specimens
Urine Specimens
[0007] The search for pathogens in urine specimens is so
preponderant in the routine microbiology laboratory that a myriad
of tests have been developed. However, the gold standard remains
the classical semi-quantitative plate culture method in which 1
.mu.L of urine is streaked on agar plates and incubated for 18-24
hours. Colonies are then counted to determine the total number of
colony forming units (CFU) per liter of urine. A bacterial urinary
tract infection (UTI) is normally associated with a bacterial count
of 10.sup.7 CFU/L or more in urine. However, infections with less
than 10.sup.7 CFU/L in urine are possible, particularly in patients
with a high incidence of diseases or those catheterized (Stark and
Maki, 1984, N. Engl. J. Med. 311:560-564). Importantly,
approximately 80% of urine specimens tested in clinical
microbiology laboratories are considered negative (i.e. bacterial
count of less than 10.sup.7 CFU/L; Table 3). Urine specimens found
positive by culture are further characterized using standard
biochemical tests to identify the bacterial pathogen and are also
tested for susceptibility to antibiotics. The biochemical and
susceptibility testing normally require 18-24 hours of
incubation.
[0008] Accurate and rapid urine screening methods for bacterial
pathogens would allow a faster identification of negative specimens
and a more efficient treatment and care management of patients.
Several rapid identification methods (Uriscreen.TM., UTIscreen.TM.,
Flash Track.TM. DNA probes and others) have been compared to slower
standard biochemical methods, which are based on culture of the
bacterial pathogens. Although much faster, these rapid tests showed
low sensitivities and poor specificities as well as a high number
of false negative and false positive results (Koening et al., 1992,
J. Clin. Microbiol. 30:342-345; Pezzlo et al., 1992, J. Clin.
Microbiol. 30:640-684).
Blood Specimens
[0009] The Blood Specimens Received In The Microbiology Laboratory
Are Always Submitted For Culture. Blood Culture Systems May Be
Manual, Semi-Automated Or Completely Automated. The BACTEC.TM.
System (From Becton Dickinson) And The Bactalert.TM. System (From
Organon Teknika Corporation) Are The Two Most Widely Used Automated
Blood Culture Systems. These Systems Incubate Blood Culture Bottles
Under Optimal Conditions For Growth Of Most Bacteria. Bacterial
Growth Is Monitored Continuously To Detect Early Positives By Using
Highly Sensitive Bacterial Growth Detectors. Once Growth Is
Detected, A Gram Stain Is Performed Directly From The Blood Culture
And Then Used To Inoculate Nutrient Agar Plates. Subsequently,
Bacterial Identification And Susceptibility Testing Are Carried Out
From Isolated Bacterial Colonies With Automated Systems As
Described Previously. Blood Culture Bottles Are Normally Reported
As Negative If No Growth Is Detected After An Incubation Of 6 To 7
Days. Normally, The Vast Majority Of Blood Cultures Are Reported
Negative. For Example, The Percentage Of Negative Blood Cultures At
The Microbiology Laboratory Of The CHUL For The Period February
1994-January 1995 Was 93.1% (Table 3).
Other Clinical Samples
[0010] Upon receipt by the clinical microbiology laboratory, all
body fluids other than blood and urine that are from normally
sterile sites (i.e. cerebrospinal, synovial, pleural, pericardial
and others) are processed for direct microscopic examination and
subsequent culture. Again, most clinical samples are negative for
culture (Table 3). In all these normally sterile sites, tests for
the universal detection of algae, archaea, bacteria, fungi and
parasites would be very useful.
[0011] Regarding clinical specimens which are not from sterile
sites such as sputum or stool specimens, the laboratory diagnosis
by culture is more problematic because of the contamination by the
normal flora. The bacterial or fungal pathogens potentially
associated with the infection are grown and separated from the
colonizing microbes using selective methods and then identified as
described previously. Of course, the DNA-based universal detection
of bacteria would not be useful for the diagnosis of bacterial
infections at these non-sterile sites. On the other hand, DNA-based
assays for species or genus or family or group detection and
identification as well as for the detection of antimicrobial agents
resistance genes from these specimens would be very useful and
would offer several advantages over classical identification and
susceptibility testing methods.
DNA-Based Assays with any Specimen
[0012] There is an obvious need for rapid and accurate diagnostic
tests for the detection and identification of algae, archaea,
bacteria, fungi and parasites directly from clinical specimens.
DNA-based technologies are rapid and accurate and offer a great
potential to improve the diagnosis of infectious diseases (Persing
et al., 1993, Diagnostic Molecular Microbiology: Principles and
Applications, American Society for Microbiology, Washington, D.C.;
Bergeron and Ouellette, 1995, Infection 23:69-72; Bergeron and
Ouellette, 1998, J Clin Microbiol. 36:2169-72). The DNA probes and
amplification primers which are objects of the present invention
are applicable for the detection and identification of algae,
archaea, bacteria, fungi, and parasites directly from any clinical
specimen such as blood, urine, sputum, cerebrospinal fluid, pus,
genital and gastro-intestinal tracts, skin or any other type of
specimens (Table 3). These assays are also applicable to detection
from microbial cultures (e.g. blood cultures, bacterial or fungal
colonies on nutrient agar, or liquid cell cultures in nutrient
broth). The DNA-based tests proposed in this invention are superior
in terms of both rapidity and accuracy to standard biochemical
methods currently used for routine diagnosis from any clinical
specimens in microbiology laboratories. Since these tests can be
performed in one hour or less, they provide the clinician with new
diagnostic tools which should contribute to a better management of
patients with infectious diseases. Specimens from sources other
than humans (e.g. other primates, birds, plants, mammals, farm
animals, livestock, food products, environment such as water or
soil, and others) may also be tested with these assays.
A High Percentage of Culture-Negative Specimens
[0013] Among all the clinical specimens received for routine
diagnosis, approximately 80% of urine specimens and even more
(around 95%) for other types of normally sterile clinical specimens
are negative for the presence of bacterial pathogens (Table 3). It
would also be desirable, in addition to identify bacteria at the
species or genus or family or group level in a given specimen, to
screen out the high proportion of negative clinical specimens with
a DNA-based test detecting the presence of any bacterium (i.e.
universal bacterial detection). As disclosed in the present
invention, such a screening test may be based on DNA amplification
by PCR of a highly conserved genetic target found in all bacteria.
Specimens negative for bacteria would not be amplified by this
assay. On the other hand, those that are positive for any bacterium
would give a positive amplification signal. Similarly, highly
conserved genes of fungi and parasites could serve not only to
identify particular species or genus or family or group but also to
detect the presence of any fungi or parasite in the specimen.
Towards the Development of Rapid DNA-Based Diagnostic Tests
[0014] A rapid diagnostic test should have a significant impact on
the management of infections. DNA probe and DNA amplification
technologies offer several advantages over conventional methods for
the identification of pathogens and antimicrobial agents resistance
genes from clinical samples (Persing et al., 1993, Diagnostic
Molecular Microbiology: Principles and Applications, American
Society for Microbiology, Washington, D.C.; Ehrlich and Greenberg,
1994, PCR-based Diagnostics in Infectious Disease, Blackwell
Scientific Publications, Boston, Mass.). There is no need for
culture of the pathogens, hence the organisms can be detected
directly from clinical samples, thereby reducing the time
associated with the isolation and identification of pathogens.
Furthermore, DNA-based assays are more accurate for microbial
identification than currently used phenotypic identification
systems which are based on biochemical tests and/or microscopic
examination. Commercially available DNA-based technologies are
currently used in clinical microbiology laboratories, mainly for
the detection and identification of fastidious bacterial pathogens
such as Mycobacterium tuberculosis, Chlamydia trachomatis,
Neisseria gonorrhoeae as well as for the detection of a variety of
viruses (Tang Y. and Persing D. H., Molecular detection and
identification of microorganisms, In: P. Murray et al., 1999,
Manual of Clinical Microbiology, ASM press, 7.sup.th edition,
Washington D.C.). There are also other commercially available
DNA-based assays which are used for culture confirmation
assays.
[0015] Others have developed DNA-based tests for the detection and
identification of bacterial pathogens which are objects of the
present invention, for example: Staphylococcus sp. (U.S. Pat. No.
5,437,978), Neisseria sp. (U.S. Pat. No. 5,162,199 and European
patent serial no. 0,337,896,131) and Listeria monocytogenes (U.S.
Pat. Nos. 5,389,513 and 5,089,386). However, the diagnostic tests
described in these patents are based either on rRNA genes or on
genetic targets different from those described in the present
invention. To our knowledge there are only four patents published
by others mentioning the use of any of the four highly conserved
gene targets described in the present invention for diagnostic
purposes (PCT international publication number WO92/03455 and
WO00/14274, European patent publication number 0 133 671 B1, and
European patent publication number 0 133 288 A2). WO92/03455 is
focused on the inhibition of Candida species for therapeutic
purposes. It describes antisense oligonucleotide probes hybridizing
to Candida messenger RNA. Two of the numerous mRNA proposed as
targets are coding for translation elongation factor 1 (tef1) and
the beta subunit of ATPase. DNA amplification or hybridization are
not under the scope of their invention and although diagnostic use
is briefly mentioned in the body of the application, no specific
claim is made regarding diagnostics. WO00/14274 describes the use
of bacterial recA gene for identification and speciation of
bacteria of the Burkholderia cepacia complex. Specific claims are
made on a method for obtaining nucleotide sequence information for
the recA gene from the target bacteria and a following comparison
with a standard library of nucleotide sequence information (claim
1), and on the use of PCR for amplification of the recA gene in a
sample of interest (claims 4 to 7, and 13). However, the use of a
discriminatory restriction enzyme in a RFLP procedure is essential
to fulfill the speciation and WO00/14274 did not mention that
multiple recA probes could be used simultaneously. Patent EP 0 133
288 A2 describes and claims the use of bacterial tuf (and fus)
sequence for diagnostics based on hybridization of a tuf (or fus)
probe with bacterial DNA. DNA amplification is not under the scope
of EP 0 133 288 A2. Nowhere it is mentioned that multiple tuf (or
fus) probes could be used simultaneously. No mention is made
regarding speciation using tuf (or fus) DNA nucleic acids and/or
sequences. The sensitivities of the tuf hybridizations reported are
1.times.10.sup.6 bacteria or 1-100 ng of DNA. This is much less
sensitive than what is achieved by our assays using nucleic acid
amplification technologies.
[0016] Although there are phenotypic identification methods which
have been used for more than 125 years in clinical microbiology
laboratories, these methods do not provide information fast enough
to be useful in the initial management of patients. There is a need
to increase the speed of the diagnosis of commonly encountered
bacterial, fungal and parasitical infections. Besides being much
faster, DNA-based diagnostic tests are more accurate than standard
biochemical tests presently used for diagnosis because the
microbial genotype (e.g. DNA level) is more stable than the
phenotype (e.g. physiologic level).
[0017] Bacteria, fungi and parasites encompass numerous well-known
microbial pathogens. Other microorganisms could also be pathogens
or associated with human diseases. For example, achlorophylious
algae of the Prototheca genus can infect humans. Archae, especially
methanogens, are present in the gut flora of humans (Reeve, J. H.,
1999, J. Bacteriol. 181:3613-3617). However, methanogens have been
associated to pathologic manifestations in the colon, vagina, and
mouth (Belay et al., 1988, Appl. Enviro. Microbiol. 54:600-603;
Belay et al., 1990, J. Clin. Microbiol. 28:1666-1668; Weaver et
al., 1986, Gut 27:698-704).
[0018] In addition to the identification of the infectious agent,
it is often desirable to identify harmful toxins and/or to monitor
the sensitivity of the microorganism to antimicrobial agents. As
revealed in this invention, genetic identification of the
microorganism could be performed simultaneously with toxin and
antimicrobial agents resistance genes. Alternatively, assays to
identify toxin and/or antimicrobial resistance genes can be
performed separately and independently from assays for
identification of infectious agents.
[0019] Knowledge of the genomic sequences of algal, archaeal,
bacterial, fungal and parasitical species continuously increases as
testified by the number of sequences available from public
databases such as GenBank. From the sequences readily available
from those public databases, there is no indication therefrom as to
their potential for diagnostic purposes. For determining good
candidates for diagnostic purposes, one could select sequences for
DNA-based assays for (i) the species-specific detection and
identification of commonly encountered bacterial, fungal and
parasitical pathogens, (ii) the genus-specific detection and
identification of commonly encountered bacterial, fungal or
parasitical pathogens, (iii) the family-specific detection and
identification of commonly encountered bacterial, fungal or
parasitical pathogens; (iv) the group-specific detection and
identification of commonly encountered bacterial, fungal or
parasitical pathogens, (v) the universal detection of algal,
archaeal, bacterial, fungal or parasitical pathogens, and/or (vi)
the specific detection and identification of antimicrobial agents
resistance genes, and/or (vii) the specific detection and
identification of bacterial toxin genes. All of the above types of
DNA-based assays may be performed directly from any type of
clinical specimens or from a microbial culture.
[0020] In our assigned U.S. Pat. No. 6,001,564 and our WO98/20157
patent publication, we described DNA sequences suitable for (i) the
species-specific detection and identification of clinically
important bacterial pathogens, (ii) the universal detection of
bacteria, and (iii) the detection of antimicrobial agents
resistance genes.
[0021] The WO98/20157 patent publication describes proprietary tuf
DNA sequences as well as tuf sequences selected from public
databases (in both cases, fragments of at least 100 base pairs), as
well as oligonucleotide probes and amplification primers derived
from these sequences. All the nucleic acid sequences described in
that patent publication can enter in the composition of diagnostic
kits or products and methods capable of a) detecting the presence
of bacteria and fungi b) detecting specifically at the species,
genus, family or group levels, the presence of bacteria and fungi
and antimicrobial agents resistance genes associated with these
pathogens. However, these methods and kits need to be improved,
since the ideal kit and method should be capable of diagnosing
close to 100% of microbial pathogens and associated antimicrobial
agents resistance genes and toxins genes. For example, infections
caused by Enterococcus faecium have become a clinical problem
because of its resistance to many antibiotics. Both the detection
of these bacteria and the evaluation of their resistance profiles
are desirable. Besides that, novel DNA sequences (probes and
primers) capable of recognizing the same and other microbial
pathogens or the same and additional antimicrobial agents
resistance genes are also desirable to aim at detecting more target
genes and complement our earlier patent applications.
[0022] The present invention improves the assigned application by
disclosing new proprietary tuf nucleic acids and/or sequences as
well as describing new ways to obtain tuf nucleic acids and/or
sequences. In addition we disclose new proprietary atpD and recA
nucleic acids and/or sequences. In addition, new uses of tuf, atpD
and recA DNA nucleic acids and/or sequences selected from public
databases (Table 11) are disclosed.
Highly Conserved Genes for Identification and Diagnostics
[0023] Highly conserved genes are useful for identification of
microorganisms. For bacteria, the most studied genes for
identification of microorganisms are the universally conserved
ribosomal RNA genes (rRNA). Among those, the principal targets used
for identification purposes are the small subunit (SSU) ribosomal
16S rRNA genes (in prokaryotes) and 18S rRNA genes (in eukaryotes)
(Relman and Persing, Genotyping Methods for Microbial
Identification, In: D. H. Persing, 1996, PCR Protocols for Emerging
Infectious Diseases, ASM Press, Washington D.C.). The rRNA genes
are also the most commonly used targets for universal detection of
bacteria (Chen et al., 1988, FEMS Microbiol. Lett. 57:19-24; McCabe
et al., 1999, Mol. Genet. Metabol. 66:205-211) and fungi (Van Burik
et al., 1998, J. Clin. Microbiol. 36:1169-1175).
[0024] However, it may be difficult to discriminate between closely
related species when using primers derived from the 16S rRNA. In
some instances, 16S rRNA sequence identity may not be sufficient to
guarantee species identity (Fox et al., 1992, Int. J. Syst.
Bacteriol. 42:166-170) and it has been shown that inter-operon
sequence variation as well as strain to strain variation could
undermine the application of 16S rRNA for identification purposes
(Clayton et al., 1995, Int. J. Syst. Bacteriol. 45:595-599). The
heat shock proteins (HSP) are another family of very conserved
proteins. These ubiquitous proteins in bacteria and eukaryotes are
expressed in answer to external stress agents. One of the most
described of these HSP is HSP 60. This protein is very conserved at
the amino acid level, hence it has been useful for phylogenetic
studies. Similar to 16S rRNA, it would be difficult to discriminate
between species using the HSP 60 nucleotide sequences as a
diagnostic tool. However, Goh et al. identified a highly conserved
region flanking a variable region in HSP 60, which led to the
design of universal primers amplifying this variable region (Goh et
al., U.S. Pat. No. 5,708,160). The sequence variations in the
resulting amplicons were found useful for the design of
species-specific assays.
SUMMARY OF THE INVENTION
[0025] Disclosed herein are compositions and methods for the
detection and quantitation of antibiotic resistant organisms from a
sample.
[0026] Some embodiments relate to compositions for the detection of
a vancomycin resistant pathogen in a sample using a nucleic acid
amplification assay. The composition can include at least one
primer pair, comprising a forward and a reverse oligonucleotide,
wherein the forward and reverse oligonucleotides each includes a
binding region that is complementary to primer binding sites
present on opposite strands of the pathogen's DNA, wherein the
primer pair is adapted to amplify a vanA or vanB gene of the
pathogen's DNA between and including said primer binding sites to
produce a detectable amplification product. The binding regions of
the oligonucleotides can correspond to, or be fully complementary
to, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
consecutive nucleotides of at least two of the following sequences:
SEQ ID NO: 1090, SEQ ID NO: 1091, SEQ ID NO: 1095, SEQ ID NO: 2298
and SEQ ID NO: 1096.
[0027] In some embodiments, the composition includes a primer pair
wherein the primers comprise, consist essentially of, or consist of
at least 10 consecutive nucleotides of SEQ ID NO: 1095 and SEQ ID
NO: 1096. Preferred compositions include a primer pair wherein the
primers comprises, consists essentially of, or consist of the
sequences of SEQ ID NO: 1095 and SEQ ID NO: 1096.
[0028] Preferably, the composition comprises at least four primers,
wherein the each of the four primers comprises, consists
essentially of, or consists of at least 10 consecutive nucleotides
of SEQ ID NOs: 1090, 1091, 1096 and 2298, or the complements
thereof. In other preferred embodiments, the four primers comprise,
consist essentially of, or consist of the SEQ ID NO: 1090, 1091,
1096, and 2298.
[0029] In some embodiments, the composition can also include at
least one internal hybridization probe, wherein the internal
hybridization probe can hybridize under stringent condition to the
vanA or vanB amplification products produced by the compositions
described above. Preferably, the internal hybridization probe is a
molecular beacon. In more preferred embodiments, the molecular
beacon can include the sequence of SEQ ID NO: 2299 or SEQ ID NO:
2300.
[0030] In further embodiments, the compositions can also include at
least one internal control DNA, which can be amplified to produce
an internal control amplicon under the same conditions and using
the same oligonucleotides of the compositions described above. In
further embodiments, the compositions can include an internal
control probe that can hybridize under stringent conditions to the
internal control amplicon. In some embodiments, the internal
control DNA comprises the sequence of SEQ ID NO: 2302. Preferably,
the internal control probe is a molecular beacon. In some
embodiments, the internal control probe includes at least 10
consecutive nucleotides of the sequence of SEQ ID NO: 2301.
[0031] Also provided herein are kits that include the compositions
described herein.
[0032] Methods to detect the presence of vancomycin-resistant
organisms in a sample are also provided. In some embodiments, the
method can include the step of annealing the nucleic acids of the
sample with at least one probe and/or primer, wherein each of the
primers and/or probes include nucleic acid sequences that
correspond to, or are fully complementary to, at least 10
consecutive nucleotides of at least two of the following sequences:
SEQ ID NO: 1090, SEQ ID NO: 1091, SEQ ID NO: 1095 and SEQ ID NO:
1096. The presence and/or amount of primer or probe that is
annealed to said sample nucleic acid can be detected. In some
embodiments, the primers and/or probes can include the nucleic acid
sequences of SEQ ID NO's 1090, 1091, 1096 and 2297, or the
complements thereof.
[0033] Preferably, the primers and/or probes are placed in the same
physical enclosure.
[0034] In some embodiments, wherein at least one pair of primers is
annealed to the sample DNA, and wherein said primer pair include
nucleic acid sequences that correspond to, or are fully
complementary to, at least 10 consecutive nucleotides of SEQ ID NO:
1090 and 1091 or SEQ ID NO: 1095 and 1096, the methods also include
a step of amplifying the sample DNA with the annealed primer
pair(s). For example, in some embodiments, the amplification step
can include a method selected from the group consisting of:
[0035] (a) polymerase chain reaction (PCR),
[0036] (b) ligase chain reaction,
[0037] (c) nucleic acid sequence-based amplification,
[0038] (d) self-sustained sequence replication,
[0039] (e) strand displacement amplification,
[0040] (f) branched DNA signal amplification,
[0041] (g) nested PCR, and
[0042] (h) multiplex PCR.
[0043] Preferably, the amplification step includes a PCR
amplification step.
[0044] In some embodiments that include an amplification step, the
sample can also be contacted with at least one probe that
hybridizes to an amplification product produced from at least one
of the primer pairs. In preferred embodiments, the probe includes
at least 10 consecutive nucleotides of the sequence of SEQ ID NO:
2299 or 2300. For example, in some embodiments, the at least one
probe includes the sequence of SEQ ID NO: 2299 or 2300.
[0045] It is an object of the present invention to provide a
specific, ubiquitous and sensitive method using probes and/or
amplification primers for determining the presence and/or amount of
nucleic acids: [0046] from any algal, archaeal, bacterial, fungal
or parasitical species in any sample suspected of containing said
nucleic acids, and optionally, [0047] from specific microbial
species or genera selected from the group consisting of the species
or genera listed in Table 4, and optionally, [0048] from an
antimicrobial agents resistance gene selected from the group
consisting of the genes listed in Table 5, and optionally, [0049]
from a toxin gene selected from the group consisting of the genes
listed in Table 6, [0050] wherein each of said nucleic acids or a
variant or part thereof comprises a selected target region
hybridizable with said probes or primers; [0051] said method
comprising the steps of contacting said sample with said probes or
primers and detecting the presence and/or amount of hybridized
probes or amplified products as an indication of the presence
and/or amount of said any microbial species, specific microbial
species or genus or family or group and antimicrobial agents
resistance gene and/or toxin gene.
[0052] In a specific embodiment, a similar method directed to each
specific microbial species or genus or family or group detection
and identification, antimicrobial agents resistance genes
detection, toxin genes detection, and universal bacterial
detection, separately, is provided.
[0053] In a more specific embodiment, the method makes use of DNA
fragments from conserved genes (proprietary sequences and sequences
obtained from public databases), selected for their capacity to
sensitively, specifically and ubiquitously detect the targeted
algal, archaeal, bacterial, fungal or parasitical nucleic
acids.
[0054] In a particularly preferred embodiment, oligonucleotides of
at least 12 nucleotides in length have been derived from the longer
DNA fragments, and are used in the present method as probes or
amplification primers. To be a good diagnostic candidate, an
oligonucleotide of at least 12 nucleotides should be capable of
hybridizing with nucleic acids from given microorganism(s), and
with substantially all strains and representatives of said
microorganism(s); said oligonucleotide being species-, or genus-,
or family-, or group-specific or universal.
[0055] In another particularly preferred embodiment,
oligonucleotides primers and probes of at least 12 nucleotides in
length are designed for their specificity and ubiquity based upon
analysis of our databases of tuf, atpD and recA sequences. These
databases are generated using both proprietary and public sequence
information. Altogether, these databases form a sequence repertory
useful for the design of primers and probes for the detection and
identification of algal, archaeal, bacterial, fungal and
parasitical microorganisms. The repertory can also be subdivided
into subrepertories for sequence analysis leading to the design of
various primers and probes.
[0056] The tuf, atpD and recA sequences databases as a product to
assist the design of oligonucleotides primers and probes for the
detection and identification of algal, archaeal, bacterial, fungal
and parasitical microorganisms are also covered.
[0057] The proprietary oligonucleotides (probes and primers) are
also another object of this invention.
[0058] Diagnostic kits comprising probes or amplification primers
such as those for the detection of a microbial species or genus or
family or phylum or group selected from the following list
consisting of Abiotrophia adiacens, Acinetobacter baumanii,
Actinomycetae, Bacteroides, Cytophaga and Flexibacter phylum,
Bacteroides fragilis, Bordetella pertussis, Bordetella sp.,
Campylobacter jejuni and C. coli, Candida albicans, Candida
dubliniensis, Candida glabrata, Candida guilliermondii, Candida
krusei, Candida lusitaniae, Candida parapsilosis, Candida
tropicalis, Candida zeylanoides, Candida sp., Chlamydia pneumoniae,
Chlamydia trachomatis, Clostridium sp., Corynebacterium sp.,
Crypococcus neoformans, Cryptococcus sp., Cryptosporidium parvum,
Entamoeba sp., Enterobacteriaceae group, Enterococcus
casseliflavus-flavescens-gallinarum group, Enterococcus faecalis,
Enterococcus faecium, Enterococcus gallinarum, Enterococcus sp.,
Escherichia coli and Shigella sp. group, Gemella sp., Giardia sp.,
Haemophilus influenzae, Klebsiella pneumoniae, Legionella
pneumophila, Legionella sp., Leishmania sp., Mycobacteriaceae
family, Mycoplasma pneumoniae, Neisseria gonorrhoeae, platelets
contaminants group (see Table 14), Pseudomonas aeruginosa,
Pseudomonads group, Staphylococcus aureus, Staphylococcus
epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis,
Staphylococcus saprophyticus, Staphylococcus sp., Streptococcus
agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes,
Streptococcus sp., Trypanosoma brucei, Trypanosoma cruzi,
Trypanosoma sp., Trypanosomatidae family, are also objects of the
present invention.
[0059] Diagnostic kits further comprising probes or amplification
primers for the detection of an antimicrobial agents resistance
gene selected from the group listed in Table 5 are also objects of
this invention.
[0060] Diagnostic kits further comprising probes or amplification
primers for the detection of a toxin gene selected from the group
listed in Table 6 are also objects of this invention.
[0061] Diagnostic kits further comprising probes or amplification
primers for the detection of any other algal, archaeal, bacterial,
fungal or parasitical species than those specifically listed
herein, comprising or not comprising those for the detection of the
specific microbial species or genus or family or group listed
above, and further comprising or not comprising probes and primers
for the antimicrobial agents resistance genes listed in Table 5,
and further comprising or not comprising probes and primers for the
toxin genes listed in Table 6 are also objects of this
invention.
[0062] In a preferred embodiment, such a kit allows for the
separate or the simultaneous detection and identification of the
above-listed microbial species or genus or family or group; or
universal detection of algae, archaea, bacteria, fungi or
parasites; or antimicrobial agents resistance genes; or toxin
genes; or for the detection of any microorganism (algae, archaea,
bacteria, fungi or parasites).
[0063] In the above methods and kits, probes and primers are not
limited to nucleic acids and may include, but are not restricted to
analogs of nucleotides such as: inosine, 3-nitropyrrole nucleosides
(Nichols et al., 1994, Nature 369:492-493), Linked Nucleic Acids
(LNA) (Koskin et al., 1998, Tetrahedron 54:3607-3630), and Peptide
Nucleic Acids (PNA) (Egholm et al., 1993, Nature 365:566-568).
[0064] In the above methods and kits, amplification reactions may
include but are not restricted to: a) polymerase chain reaction
(PCR), b) ligase chain reaction (LCR), c) nucleic acid
sequence-based amplification (NASBA), d) self-sustained sequence
replication (3SR), e) strand displacement amplification (SDA), f)
branched DNA signal amplification (bDNA), g) transcription-mediated
amplification (TMA), h) cycling probe technology (CPT), i) nested
PCR, j) multiplex PCR, k) solid phase amplification (SPA), 1)
nuclease dependent signal amplification (NDSA), m) rolling circle
amplification technology (RCA), n) Anchored strand displacement
amplification, o) Solid-phase (immobilized) rolling circle
amplification.
[0065] In the above methods and kits, detection of the nucleic
acids of target genes may include real-time or post-amplification
technologies. These detection technologies can include, but are not
limited to, fluorescence resonance energy transfer (FRET)-based
methods such as adjacent hybridization to FRET probes (including
probe-probe and probe-primer methods), TaqMan, Molecular Beacons,
scorpions, nanoparticle probes and Sunrise (Amplifluor). Other
detection methods include target genes nucleic acids detection via
immunological methods, solid phase hybridization methods on
filters, chips or any other solid support, whether the
hybridization is monitored by fluorescence, chemiluminescence,
potentiometry, mass spectrometry, plasmon resonance, polarimetry,
colorimetry, or scanometry. Sequencing, including sequencing by
dideoxy termination or sequencing by hybridization, e.g. sequencing
using a DNA chip, is another possible method to detect and identify
the nucleic acids of target genes.
[0066] In a preferred embodiment, a PCR protocol is used for
nucleic acid amplification, in diagnostic method as well as in
method of construction of a repertory of nucleic acids and deduced
sequences.
[0067] In a particularly preferred embodiment, a PCR protocol is
provided, comprising, an initial denaturation step of 1-3 minutes
at 95.degree. C., followed by an amplification cycle including a
denaturation step of one second at 95.degree. C. and an annealing
step of 30 seconds at 45-65.degree. C., without any time allowed
specifically for the elongation step. This PCR protocol has been
standardized to be suitable for PCR reactions with most selected
primer pairs, which greatly facilitates the testing because each
clinical sample can be tested with universal, species-specific,
genus-specific, antimicrobial agents resistance gene and toxin gene
PCR primers under uniform cycling conditions. Furthermore, various
combinations of primer pairs may be used in multiplex PCR
assays.
[0068] It is also an object of the present invention that tuf, atpD
and recA sequences could serve as drug targets and these sequences
and means to obtain them revealed in the present invention can
assist the screening, design and modeling of these drugs.
[0069] It is also an object of the present invention that tuf, atpD
and recA sequences could serve for vaccine purposes and these
sequences and means to obtain them revealed in the present
invention can assist the screening, design and modeling of these
vaccines.
[0070] We aim at developing a universal DNA-based test or kit to
screen out rapidly samples which are free of algal, archaeal,
bacterial, fungal or parasitical cells. This test could be used
alone or combined with more specific identification tests to detect
and identify the above algal and/or archaeal and/or bacterial
and/or fungal and/or parasitical species and/or genera and/or
family and/or group and to determine rapidly the bacterial
resistance to antibiotics and/or presence of bacterial toxins.
Although the sequences from the selected antimicrobial agents
resistance genes are available from public databases and have been
used to develop DNA-based tests for their detection, our approach
is unique because it represents a major improvement over current
diagnostic methods based on bacterial cultures. Using an
amplification method for the simultaneous or independent or
sequential microbial detection-identification and antimicrobial
resistance genes detection, there is no need for culturing the
clinical sample prior to testing. Moreover, a modified PCR protocol
has been developed to detect all target DNA sequences in
approximately one hour under uniform amplification conditions. This
procedure should save lives by optimizing treatment, should
diminish antimicrobial agents resistance because less antibiotics
will be prescribed, should reduce the use of broad spectrum
antibiotics which are expensive, decrease overall health care costs
by preventing or shortening hospitalizations, and side effects of
drugs, and decrease the time and costs associated with clinical
laboratory testing.
[0071] In another embodiment, sequence repertories and ways to
obtain them for other gene targets are also an object of this
invention, such is the case for the hexA nucleic acids and/or
sequences of Streptococci.
[0072] In yet another embodiment, for the detection of mutations
associated with antibiotic resistance genes, we built repertories
to distinguish between point mutations reflecting only gene
diversity and point mutations involved in resistance. Such
repertories and ways to obtain them for pbp1a, pbp2b and pbp2x
genes of sensitive and penicillin-resistant Streptococcus
pneumoniae and also for gyrA and parC gene fragments from various
bacterial species are also an object of the present invention.
[0073] The diagnostic kits, primers and probes mentioned above can
be used to identify algae, archaea, bacteria, fungi, parasites,
antimicrobial agents resistance genes and toxin genes on any type
of sample, whether said diagnostic kits, primers and probes are
used for in vitro or in situ applications. The said samples may
include but are not limited to: any clinical sample, any
environment sample, any microbial culture, any microbial colony,
any tissue, and any cell line.
[0074] It is also an object of the present invention that said
diagnostic kits, primers and probes can be used alone or in
conjunction with any other assay suitable to identify
microorganisms, including but not limited to: any immunoassay, any
enzymatic assay, any biochemical assay, any lysotypic assay, any
serological assay, any differential culture medium, any enrichment
culture medium, any selective culture medium, any specific assay
medium, any identification culture medium, any enumeration culture
medium, any cellular stain, any culture on specific cell lines, and
any infectivity assay on animals.
[0075] In the methods and kits described herein below, the
oligonucleotide probes and amplification primers have been derived
from larger sequences (i.e. DNA fragments of at least 100 base
pairs). All DNA fragments have been obtained either from
proprietary fragments or from public databases. DNA fragments
selected from public databases are newly used in a method of
detection according to the present invention, since they have been
selected for their diagnostic potential.
[0076] In another embodiment, the amino acid sequences translated
from the repertory of tuf, atpD and recA nucleic acids and/or
sequences are also an object of the present invention.
[0077] It is clear to the individual skilled in the art that other
oligonucleotide sequences appropriate for (i) the universal
detection of algae, archaea, bacteria, fungi or parasites, (ii) the
detection and identification of the above microbial species or
genus or family or group, and (iii) the detection of antimicrobial
agents resistance genes, and (iv) the detection of toxin genes,
other than those listed in Annexes I to III, XXI to XXII, XXXII to
XXXVII, XXXIX to XLI, and XLIII to LIV may also be derived from the
proprietary fragments or selected public database sequences. For
example, the oligonucleotide primers or probes may be shorter or
longer than the ones chosen; they may also be selected anywhere
else in the proprietary DNA fragments or in the sequences selected
from public databases; they may be also variants of the same
oligonucleotide. If the target DNA or a variant thereof hybridizes
to a given oligonucleotide, or if the target DNA or a variant
thereof can be amplified by a given oligonucleotide PCR primer
pair, the converse is also true; a given target DNA may hybridize
to a variant oligonucleotide probe or be amplified by a variant
oligonucleotide PCR primer. Alternatively, the oligonucleotides may
be designed from any DNA fragment sequences for use in
amplification methods other than PCR. Consequently, the core of
this invention is the identification of universal,
species-specific, genus-specific, family-specific, group-specific,
resistance gene-specific, toxin gene-specific genomic or
non-genomic DNA fragments which are used as a source of specific
and ubiquitous oligonucleotide probes and/or amplification primers.
Although the selection and evaluation of oligonucleotides suitable
for diagnostic purposes requires much effort, it is quite possible
for the individual skilled in the art to derive, from the selected
DNA fragments, oligonucleotides other than the ones listed in
Annexes I to III, XXI to XXII, XXXII to XXXVII, XXXIX to XLI, and
XLIII to LIV which are suitable for diagnostic purposes. When a
proprietary fragment or a public databases sequence is selected for
its specificity and ubiquity, it increases the probability that
subsets thereof will also be specific and ubiquitous.
[0078] Since a high percentage of clinical specimens are negative
for bacteria (Table 3), DNA fragments having a high potential for
the selection of universal oligonucleotide probes or primers were
selected from proprietary and public database sequences. The
amplification primers were selected from genes highly conserved in
algae, archaea, bacteria, fungi and parasites, and are used to
detect the presence of any algal, archaeal, bacterial, fungal or
parasitical pathogen in clinical specimens in order to determine
rapidly whether it is positive or negative for algae, archaea,
bacteria, fungi or parasites. The selected genes, designated tuf,
fus, atpD and recA, encode respectively 2 proteins (elongation
factors Tu and G) involved in the translational process during
protein synthesis, a protein (beta subunit) responsible for the
catalytic activity of proton pump ATPase and a protein responsible
for the homologous recombination of genetic material. The
alignments of tuf, atpD and recA sequences used to derive the
universal primers include both proprietary and public database
sequences. The universal primer strategy allows the rapid screening
of the numerous negative clinical specimens (around 80% of the
specimens received, see Table 3) submitted for microbiological
testing.
[0079] Table 4 provides a list of the archaeal, bacterial, fungal
and parasitical species for which tuf and/or atpD and/or recA
nucleic acids and/or sequences are revealed in the present
invention. Tables 5 and 6 provide a list of antimicrobial agents
resistance genes and toxin genes selected for diagnostic purposes.
Table 7 provides the origin of tuf, atpD and recA nucleic acids
and/or sequences listed in the sequence listing. Tables 8-10 and
12-14 provide lists of species used to test the specificity,
ubiquity and sensitivity of some assays described in the examples.
Table 11 provides a list of microbial species for which tuf and/or
atpD and/or recA sequences are available in public databases. Table
15 lists the microorganisms identified by commercial systems.
Tables 16-18 are part of Example 42, whereas Tables 19-20 are part
of Example 43. Tables 21-22 illustrate Example 44, whereas Tables
23-25 illustrate Example 45.
BRIEF DESCRIPTION OF THE DRAWINGS
[0080] FIGS. 1 and 2 illustrate the principal subdivisions of the
tuf and atpD sequences repertories, respectively. For the design of
primers and probes, depending on the needs, one may want to use the
complete data set illustrated on the top of the pyramid or use only
a subset illustrated by the different branching points. Smaller
subdivisions, representing groups, families, genus and species,
could even be made to extend to the bottom of the pyramid. Because
the tuf and atpD sequences are highly conserved and evolved with
each species, the design of primers and probes does not need to
include all the sequences within the database or its subdivisions.
As illustrated in Annexes IV to XX, XXIII to XXXI, XXXVIII and
XLII, depending on the use, sequences from a limited number of
species can be carefully selected to represent: i) only the main
phylogenetic branches from which the intended probes and primers
need to be differentiating, and ii) only the species for which they
need to be matching. However, for ubiquity purposes, and especially
for primers and probes identifying large groups of species (genus,
family, group or universal, or sequencing primers), the more data
is included into the sequence analysis, the better the probes and
primers will be suitable for each particular intended use.
Similarly, for specificity purposes, a larger data set (or
repertory) ensures optimal primers and probes design by reducing
the chance of employing nonspecific oligonucleotides.
[0081] FIG. 3 illustrates the approach used to design specific
amplification primers from fusA as well as from the region between
the end of fusA and the beginning of tuf in the streptomycin (str)
operon (referred to as the fusA-tuf intergenic spacer in Table 7).
Shown is a schematic organization of universal amplification
primers (SEQ ID NOs. 1221-1229) in the str operon. Amplicon sizes
are given in bases pairs. Drawing not to scale, as the fusA-tuf
intergenic spacer size varies depending on the bacterial species.
Indicated amplicon lengths are for E. coli.
[0082] FIGS. 4 to 6 are illustrations to Example 42, whereas FIGS.
7 to 10 illustrate Example 43. FIGS. 11 and 12 illustrate Example
44.
[0083] FIG. 4. Abridged multiple amino acid sequence alignment of
the partial tuf gene products from selected species illustrated
using the program Alscript. Residues highly conserved in bacteria
are boxed in grey and gaps are represented with dots. Residues in
reverse print are unique to the enterococcal tufB as well as to
streptococcal and lactococcal tuf gene products. Numbering is based
on E. coli EF-Tu and secondary structure elements of E. coli EF-Tu
are represented by cylinders (.alpha.-helices) and arrows
(.beta.-strands).
[0084] FIG. 5. Distance matrix tree of bacterial EF-Tu based on
amino acid sequence homology. The tree was constructed by the
neighbor-joining method. The tree was rooted using archeal and
eukaryotic EF-1.alpha. genes as the outgroup. The scale bar
represents 5% changes in amino acid sequence, as determined by
taking the sum of all of the horizontal lines connecting two
species.
[0085] FIG. 6. Southern hybridization of BglII/XbaI digested
genomic DNAs of some enterococci (except for E. casseliflavus and
E. gallinarum whose genomic DNA was digested with BamHI/PvuII)
using the tufA gene fragment of E. faecium as probes. The sizes of
hybridizing fragments are shown in kilobases. Strains tested are
listed in Table 16.
[0086] FIG. 7. Pantoea and Tatumella species specific signature
indel in atpD genes. The nucleotide positions given are for E. coli
atpD sequence (GenBank accession no. V00267). Numbering starts from
the first base of the initiation codon.
[0087] FIG. 8: Trees based on sequence data from tuf (left side)
and atpD (right side). The phylogenetic analysis was performed
using the Neighbor-Joining method calculated using the Kimura
two-parameter method. The value on each branch indicates the
occurrence (%) of the branching order in 750 bootstrapped
trees.
[0088] FIG. 9: Phylogenetic tree of members of the family
Enterobacteriaceae based on tuf (a), atpD (b), and 16S rDNA (c)
genes. Trees were generated by neighbor-joining method calculated
using the Kimura two-parameter method. The value on each branch is
the percentage of bootstrap replications supporting the branch. 750
bootstrap replications were calculated.
[0089] FIG. 10: Plot of tuf distances versus 16S rDNA distances
(a), atpD distances versus 16S rDNA distances (b), and atpD
distances versus tuf distances (c). Symbols: .largecircle.,
distances between pairs of strains belonging to the same species; ,
distances between E. coli strains and Shigella strains;
.quadrature., distances between pairs belonging to the same genus;
.box-solid., distances between pairs belonging to different genera;
.DELTA., distances between pairs belonging to different
families.
[0090] FIG. 11 depicts a multiple nucleic acid sequence alignment
of the vanA gene from the indicated GenBank nucleotide accession
numbers. Above the alignment of the GenBank sequences is a
consensus sequence, derived from the alignment of the nucleic acid
sequences below. Below the alignment of the GenBank sequences and
shaded in grey are the sequences of oligonucleotides (SEQ ID NOs:
1090 and 1091) and the position of a molecular beacon probe (SEQ ID
NO: 2299) that hybridizes to the amplification product of SEQ ID
NOs: 1090 and 1091.
[0091] FIG. 12 depicts a multiple nucleic acid sequence alignment
of the vanB gene from the indicated GenBank nucleotide accession
numbers. Above the alignment of the GenBank sequences is a
consensus sequence, derived from the alignment of the nucleic acid
sequences below. Below the alignment of the GenBank sequences and
shaded in grey are the sequences of oligonucleotides (SEQ ID NOs:
1096 and 2298) and the position of a molecular beacon probe (SEQ ID
NO: 2300) that hybridizes to the amplification product of SEQ ID
NOs: 1096 and 2298.
[0092] FIGS. 13A and 13B shows a graphical depictions of PCR
amplification curves measured from reactions containing molecular
beacon probes. Reactions contained 0, 0.5, 2.5, 5. 10, or 20 copies
of vanA resistant E. faecium (FIG. 13A) or vanB resistant E.
faecalis (FIG. 13B) template DNA, as well as 3.5 copies of internal
control DNA. Molecular beacon probes (SEQ ID NO: 2299 and 2300)
were added to each reaction and the fluorescence of the reactions
was measured (FIGS. 13A and 13B, respectively). SEQ ID NO: 2299 is
labeled with FAM. SEQ ID NO: 2300 is labeled with Texas Red. SEQ ID
NO: 2301 is labeled with TET.
[0093] FIG. 14 shows an agarose gel of the DNA amplification
products from PCR using the template DNA sources listed in Table
29. The numbers above the lanes correspond to the numbers in Table
29.
[0094] FIG. 15 shows an agarose gel of the DNA amplification
products from PCR using template DNA sources listed in Table 30.
The numbers above the lanes correspond to the numbers in Table
30.
[0095] FIG. 16 shows an agarose gel of the DNA amplification
products from PCR using template DNA sources listed in Table 31.
The numbers above the lanes correspond to the numbers in Table
31.
[0096] FIG. 17 shows an agarose gel of the DNA amplification
products from PCR using template DNA sources listed in Table 32.
The numbers above the lanes correspond to the numbers in Table
32.
[0097] FIGS. 18A and 18B show the fluorescence signal readout
obtained in the FAM channel when vanA template (FIG. 18A) or
non-specific template (FIG. 18B) DNA was used in PCR according to
Example 23.
[0098] FIGS. 19A and 19B show the fluorescence signal readout
obtained in the TET channel when internal control (IC) template
(FIG. 19A) or non-specific template (FIG. 19B) DNA was used in PCR
according to Example 23
[0099] FIGS. 20A and 20B show the fluorescence signal readout
obtained in the Texas Red channel when vanB template (FIG. 20A) or
non-specific template (FIG. 20B) DNA was used in the PCR according
to Example 23.
[0100] FIG. 21 shows the fluorescent signal readout obtained in the
vanR assay for a vanA positive clinical specimen. The top panel
shows the fluorescent readout from the FAM channel, and the bottom
panel shows the fluorescent readout in the Texas Red channel,
designed to detect the vanB probe.
[0101] FIG. 22 shows the fluorescent signal readout obtained in the
vanR assay for a clinical specimen that is both vanA and vanB
positive. The top panel shows the readout from the FAM channel
(vanA) and the bottom panel a shows the readout from the Texas Red
channel (vanB).
[0102] FIG. 23 shows the fluorescent signal readout obtained in the
vanR assay for a clinical specimen that is vanB positive. The top
panel shows the FAM channel (vanA) and the bottom channel shows the
fluorescent readout from the Texas Red channel (vanB).
[0103] FIG. 24 shows an agarose gel of the DNA amplification
products from PCR using the template DNA sources listed in Table
36. The numbers above the lanes correspond to the numbers in Table
36.
[0104] FIG. 25 shows an agarose gel of the DNA amplification
products from PCR using the template DNA sources listed in Table
37. The numbers above the lanes correspond to the numbers in Table
37.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0105] The present inventors reasoned that comparing the published
Haemophilus influenzae and Mycoplasma genitalium genomes and
searching for conserved genes could provide targets to develop
useful diagnostic primers and probes. This sequence comparison is
highly informative as these two bacteria are distantly related and
most genes present in the minimal genome of M. genitalium are
likely to be present in every bacterium. Therefore genes conserved
between these two bacteria are likely to be conserved in all other
bacteria.
[0106] Following the genomic comparison, it was found that several
protein-coding genes were conserved in evolution. Highly conserved
proteins included the translation elongation factors G (EF-G) and
Tu (EF-Tu) and the .beta. subunit of F.sub.0F.sub.1 type
ATP-synthase, and to a lesser extent, the RecA recombinase. These
four proteins coding genes were selected amongst the 20 most
conserved genes on the basis that they all possess at least two
highly conserved regions suitable for the design of universal
amplification and sequencing primers. Moreover, within the fragment
amplified by these primers, highly conserved and more variable
regions are also present hence suggesting it might be possible to
rapidly obtain sequence information from various microbial species
to design universal as well as species-, genus-, family-, or
group-specific primers and probes of potential use for the
detection and identification and/or quantification of
microorganisms.
[0107] Translation elongation factors are members of a family of
GTP-binding proteins which intervene in the interactions of tRNA
molecules with the ribosome machinery during essential steps of
protein synthesis. The role of elongation factor Tu is to
facilitate the binding of aminoacylated tRNA molecules to the A
site of the ribosome. The eukaryotic, archaeal (archaebacterial)
and algal homolog of EF-Tu is called elongation factor 1 alpha
(EF-1.alpha.). All protein synthesis factors originated from a
common ancestor via gene duplications and fusions (Cousineau et
al., 1997, J. Mol. Evol. 45:661-670). In particular, elongation
factor G (EF-G), although having a functional role in promoting the
translocation of aminoacyl-tRNA molecules from the A site to the P
site of the ribosome, shares sequence homologies with EF-Tu and is
thought to have arisen from the duplication and fusion of an
ancestor of the EF-Tu gene.
[0108] In addition, EF-Tu is known to be the target for antibiotics
belonging to the elfamycin's group as well as to other structural
classes (Anborgh and Parmeggiani, 1991, EMBO J. 10:779-784; Luiten
et al., 1992, European patent application serial No. EP 0 466 251
A1). EF-G for its part, is the target of the antibiotic fusidic
acid. In addition to its crucial activities in translation, EF-Tu
has chaperone-like functions in protein folding, protection against
heat denaturation of proteins and interactions with unfolded
proteins (Caldas et al., 1998, J. Biol. Chem 273:11478-11482).
Interestingly, a form of the EF-Tu protein has been identified as a
dominant component of the periplasm of Neisseria gonorrhoeae
(Porcella et al., 1996, Microbiology 142:2481-2489), hence
suggesting that at least in some bacterial species, EF-Tu might be
an antigen with vaccine potential.
[0109] F.sub.0F.sub.1 type ATP-synthase belongs to a superfamily of
proton-translocating ATPases divided in three major families: P, V
and F (Nelson and Taiz, 1989, TIBS 14:113-116). P-ATPases (or
E.sub.1-E.sub.2 type) operate via a phosphorylated intermediate and
are not evolutionarily related to the other two families. V-ATPases
(or V.sub.0V.sub.1 type) are present on the vacuolar and other
endomembranes of eukaryotes, on the plasma membrane of archaea
(archaebacteria) and algae, and also on the plasma membrane of some
eubacteria especially species belonging to the order Spirochaetales
as well as to the Chlamydiaceae and Deinococcaceae families.
F-ATPases (or F.sub.0F.sub.1 type) are found on the plasma membrane
of most eubacteria, on the inner membrane of mitochondria and on
the thylakoid membrane of chloroplasts. They function mainly in ATP
synthesis. They are large multimeric enzymes sharing numerous
structural and functional features with the V-ATPases. F and V-type
ATPases have diverged from a common ancestor in an event preceding
the appearance of eukaryotes. The .beta. subunit of the F-ATPases
is the catalytic subunit and it possesses low but significant
sequence homologies with the catalytic A subunit of V-ATPases.
[0110] The translation elongation factors EF-Tu, EF-G and
EF-1.alpha. and the catalytic subunit of F or V-types ATP-synthase,
are highly conserved proteins sometimes used for phylogenetic
analysis and their genes are also known to be highly conserved
(Iwabe et al., 1989, Proc. Natl. Acad. Sci. USA 86:9355-9359,
Gogarten et al., 1989, Proc. Natl. Acad. Sci. USA 86:6661-6665,
Ludwig et al., 1993, Antonie van Leeuwenhoek 64:285-305).
[0111] A recent BLAST (Altschul et al., 1997, J. Mol. Biol.
215:403-410) search performed by the present inventors on the
GenBank, European Molecular Biology Laboratory (EMBL), DNA Database
of Japan (DDBJ) and specific genome project databases indicated
that throughout bacteria, the EF-Tu and the .beta. subunit of
F.sub.0F.sub.1 type ATP-synthase genes may be more conserved than
other genes that are well conserved between H. influenzae and M.
genitalium.
[0112] The RecA recombinase is a multifunctional protein encoded by
the recA gene. It plays a central role in homologous recombination,
it is critical for the repair of DNA damage and it is involved in
the regulation of the SOS system by promoting the proteolytic
digestion of the LexA repressor. It is highly conserved in bacteria
and could serve as a useful genetic marker to reconstruct bacterial
phylogeny (Miller and Kokjohn, 1990, Annu. Rev. Microbiol.
44:365-394). Although RecA possesses some highly conserved sequence
segments that we used to design universal primers aimed at
sequencing the recA fragments, it is clearly not as well conserved
EF-G, EF-Tu and (3 subunit of F.sub.0F.sub.1 type ATP-synthase.
Hence, RecA may not be optimal for universal detection of bacteria
with high sensitivity but it was chosen because preliminary data
indicated that EF-G, EF-Tu and 1 subunit of F.sub.0F.sub.1 type
ATP-synthase may sometimes be too closely related to find specific
primer pairs that could discriminate between certain very closely
related species and genera. While RecA, EF-G, EF-Tu and .beta.
subunit of F.sub.0F.sub.1 type ATP-synthase genes, possesses highly
conserved regions suitable for the design of universal sequencing
primers, the less conserved region between primers should be
divergent enough to allow species-specific and genus-specific
primers in those cases.
[0113] Thus, as targets to design primers and probes for the
genetic detection of microorganisms, the present inventors have
focused on the genes encoding these four proteins: tuf, the gene
for elongation factor Tu (EF-Tu); fus, the gene for the elongation
factor G (EF-G); atpD, the gene for .beta. subunit of
F.sub.0F.sub.1 type ATP-synthase; and recA, the gene encoding the
RecA recombinase. In several bacterial genomes tuf is often found
in two highly similar duplicated copies named tufA and tufB (Filer
and Furano, 1981, J. Bacteriol. 148:1006-1011, Sela et al., 1989,
J. Bacteriol. 171:581-584). In some particular cases, more
divergent copies of the tuf genes can exist in some bacterial
species such as some actinomycetes (Luiten et al. European patent
application publication No. EP 0 446 251 A 1; Vijgenboom et al.,
1994, Microbiology 140:983-998) and, as revealed as part of this
invention, in several enterococcal species. In several bacterial
species, tuf is organized in an operon with its homolog gene for
the elongation factor G (EF-G) encoded by the fusA gene (FIG. 3).
This operon is often named the str operon. The tuf, fus, atpD and
recA genes were chosen as they are well conserved in evolution and
have highly conserved stretches as well as more variable segments.
Moreover, these four genes have eukaryotic orthologs which are
described in the present invention as targets to identify fungi and
parasites. The eukaryotic homolog of elongation factor Tu is called
elongation factor 1-alpha (EF-1.alpha.) (gene name: tef, tef1, ef1,
ef-1 or EF-1). In fungi, the gene for EF-1.alpha. occurs sometimes
in two or more highly similar duplicated copies (often named tef1,
tef 2, tef3 . . . ). In addition, eukaryotes have a copy of
elongation factor Tu which is originating from their organelle
genome ancestry (gene name: tuf1, tufM or tufA). For the purpose of
the current invention, the genes for these four functionally and
evolutionarily linked elongation factors (bacterial EF-Tu and EF-G,
eukaryotic EF-1.alpha., and organellar EF-Tu) will hereafter be
designated as <<tuf nucleic acids and/or sequences>>.
The eukaryotic (mitochondrial) F.sub.0F.sub.1 type ATP-synthase
beta subunit gene is named atp2 in yeast. For the purpose of the
current invention, the genes of catalytic sub-unit of either F or
V-type ATP-synthase will hereafter be designated as <<atpD
nucleic acids and/or sequences>>. The eukaryotic homologs of
RecA are distributed in two families, typified by the Rad51 and
Dmc1 proteins. Archaeal homologs of RecA are called RadA. For the
purpose of the current invention, the genes corresponding to the
latter proteins will hereafter be designated as <<recA
nucleic acids and/or sequences>>.
[0114] In the description of this invention, the terms
<<nucleic acids>> and <<sequences>> might
be used interchangeably. However, <<nucleic acids>> are
chemical entities while <<sequences>> are the pieces of
information derived from (inherent to) these <<nucleic
acids>>. Both nucleic acids and sequences are equivalently
valuable sources of information for the matter pertaining to this
invention.
[0115] Analysis of multiple sequence alignments of tuf and atpD
sequences permitted the design of oligonucleotide primers (and
probes) capable of amplifying (or hybridizing to) segments of tuf
(and/or fus) and atpD genes from a wide variety of bacterial
species (see Examples 1 to 4, 24 and 26, and Table 7). Sequencing
and amplification primer pairs for tuf nucleic acids and/or
sequences are listed in Annex I and hybridization probes are listed
in Annexes III and XLVII. Sequencing and amplification primer pairs
for atpD nucleic acids and/or sequences are listed in Annex II.
Analysis of the main subdivisions of tuf and atpD sequences (see
FIGS. 1 and 2) permitted to design sequencing primers amplifying
specifically each of these subdivisions. It should be noted that
these sequencing primers could also be used as universal primers.
However, since some of these sequencing primers include several
variable sequence (degenerated) positions, their sensitivity could
be lower than that of universal primers developed for diagnostic
purposes. Further subdivisions could be done on the basis of the
various phyla where these genes are encountered.
[0116] Similarly, analysis of multiple sequence alignments of recA
sequences present in the public databases permitted the design of
oligonucleotide primers capable of amplifying segments of recA
genes from a wide variety of bacterial species. Sequencing and
amplification primer pairs for recA sequences are listed in Annex
XXI. The main subdivisions of recA nucleic acids and/or sequences
comprise recA, radA, rad51 and dmc1. Further subdivisions could be
done on the basis of the various phyla where these genes are
encountered.
[0117] The present inventor's strategy is to get as much sequence
data information from the four conserved genes (tuf, fus, atpD and
recA). This ensemble of sequence data forming a repertory (with
subrepertories corresponding to each target gene and their main
sequence subdivisions) and then using the sequence information of
the sequence repertory (or subrepertories) to design primer pairs
that could permit either universal detection of algae or archaea or
bacteria or fungi or parasites, detection of a family or group of
microorganism (e.g. Enterobacteriaceae), detection of a genus (e.g.
Streptococcus) or finally a specific species (e.g. Staphylococcus
aureus). It should be noted that for the purpose of the present
invention a group of microorganisms is defined depending on the
needs of the particular diagnostic test. It does not need to
respect a particular taxonomical grouping or phylum. See Example 12
where primers were designed to amplify a group a bacteria
consisting of the 17 major bacterial species encountered as
contaminants of platelet concentrates. Also remark that in that
Example, the primers are not only able to sensitively and rapidly
detect at least the 17 important bacterial species, but could also
detect other species as well, as shown in Table 14. In these
circumstances the primers shown in Example 12 are considered
universal for platelet-contaminating bacteria. To develop an assay
specific for the latter, one or more primers or probes specific to
each species could be designed. Another example of primers and/or
probes for group detection is given by the Pseudomonad group
primers. These primers were designed based upon alignment of tuf
sequences from real Pseudomonas species as well as from former
Pseudomonas species such as Stenotrophomonas maltophilia. The
resulting primers are able to amplify all Pseudomonas species
tested as well as several species belonging to different genera,
hence as being specific for a group including Pseudomonas and other
species, we defined that group as Pseudomonads, as several members
were former Pseudomonas.
[0118] For certain applications, it may be possible to develop a
universal, group, family or genus-specific reaction and to proceed
to species identification using sequence information within the
amplicon to design species-specific internal probes or primers, or
alternatively, to proceed directly by sequencing the amplicon. The
various strategies will be discussed further below.
[0119] The ensembles formed by public and proprietary tuf, atpD and
recA nucleic acids and/or sequences are used in a novel fashion so
they constitute three databases containing useful information for
the identification of microorganisms.
[0120] Sequence repertories of other gene targets were also built
to solve some specific identification problems especially for
microbial species genetically very similar to each other such as E.
coli and Shigella (see Example 23). Based on tuf, atpD and recA
sequences, Streptococcus pneumoniae is very difficult to
differentiate from the closely related species S. oralis and S.
mitis. Therefore, we elected to built a sequence repertory from
hexA sequences (Example 19), a gene much more variable than our
highly conserved tuf, atpD and recA nucleic acids and/or
sequences.
[0121] For the detection of mutations associated with antibiotic
resistance genes, we also built repertories to distinguish between
point mutations reflecting only gene diversity and point mutations
involved in resistance. This was done for pbp1a, pbp2b and pbp2x
genes of penicillin-resistant and sensitive Streptococcus
pneumoniae (Example 18) and also for gyrA and parC gene fragments
of various bacterial species for which quinolone resistance is
important to monitor.
Oligonucleotide Primers and Probes Design and Synthesis
[0122] The tuf, fus, atpD and recA DNA fragments sequenced by us
and/or selected from public databases (GenBank and EMBL) were used
to design oligonucleotides primers and probes for diagnostic
purposes. Multiple sequence alignments were made using subsets of
the tuf or atpD or recA sequences repertory. Subsets were chosen to
encompass as much as possible of the targeted microorganism(s) DNA
sequence data and also include sequence data from phylogenetically
related microorganisms from which the targeted microorganism(s)
should be distinguished. Regions suitable for primers and probes
should be conserved for the targeted microorganism(s) and divergent
for the microorganisms from which the targeted microorganism(s)
should be distinguished. The large amount of tuf or atpD or recA
sequences data in our repertory permits to reduce trial and errors
in obtaining specific and ubiquitous primers and probes. We also
relied on the corresponding peptide sequences of tuf, fus, atpD and
recA nucleic acids and/or sequences to facilitate the
identification of regions suitable for primers and probes design.
As part of the design rules, all oligonucleotides (probes for
hybridization and primers for DNA amplification by PCR) were
evaluated for their suitability for hybridization or PCR
amplification by computer analysis using standard programs (i.e.
the Genetics Computer Group (GCG) programs and the primer analysis
software Oligo.TM. 5.0). The potential suitability of the PCR
primer pairs was also evaluated prior to the synthesis by verifying
the absence of unwanted features such as long stretches of one
nucleotide and a high proportion of G or C residues at the 3' end
(Persing et al., 1993, Diagnostic Molecular Microbiology:
Principles and Applications, American Society for Microbiology,
Washington, D.C.). Oligonucleotide probes and amplification primers
were synthesized using an automated DNA synthesizer (Perkin-Elmer
Corp., Applied Biosystems Division).
[0123] The oligonucleotide sequence of primers or probes may be
derived from either strand of the duplex DNA. The primers or probes
may consist of the bases A, G, C, or T or analogs and they may be
degenerated at one or more chosen nucleotide position(s). The
primers or probes may be of any suitable length and may be selected
anywhere within the DNA sequences from proprietary fragments or
from selected database sequences which are suitable for (i) the
universal detection of algae or archaea or bacteria or fungi or
parasites, (ii) the species-specific detection and identification
of any microorganism, including but not limited to: Abiotrophia
adiacens, Bacteroides fragilis, Bordetella pertussis, Candida
albicans, Candida dubliniensis, Candida glabrata, Candida
guilliermondii, Candida krusei, Candida lusitaniae, Candida
parapsilosis, Candida tropicalis, Candida zeylanoides,
Campylobacter jejuni and C. coli, Chlamydia pneumoniae, Chlamydia
trachomatis, Cryptococcus neoformans, Cryptosporidium parvum,
Enterococcus faecalis, Enterococcus faecium, Enterococcus
gallinarum, Escherichia coli, Haemophilus influenzae, Legionella
pneumophila, Mycoplasma pneumoniae, Neisseria gonorrhoeae,
Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus
epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis,
Staphylococcus saprophyticus, Streptococcus agalactiae,
Streptococcus pneumoniae, Trypanosoma brucei, Trypanosoma cruzi,
(iii) the genus-specific detection of Bordetella species, Candida
species, Clostridium species, Corynebacterium species, Cryptococcus
species, Entamoeba species, Enterococcus species, Gemella species,
Giardia species, Legionella species, Leishmania species,
Staphylococcus species, Streptococcus species, Trypanosoma species,
(iv) the family-specific detection of Enterobacteriaceae family
members, Mycobacteriaceae family members, Trypanosomatidae family
members, (v) the detection of Enterococcus
casseliflavus-flavescens-gallinarum group, Enterococcus, Gemella
and Abiotrophia adiacens group, Pseudomonads extended group,
Platelet-contaminating bacteria group, (vi) the detection of
clinically important antimicrobial agents resistance genes listed
in Table 5, (vii) the detection of clinically important toxin genes
listed in Table 6.
[0124] Variants for a given target microbial gene are naturally
occurring and are attributable to sequence variation within that
gene during evolution (Watson et al., 1987, Molecular Biology of
the Gene, 4.sup.th ed., The Benjamin/Cummings Publishing Company,
Menlo Park, Calif.; Lewin, 1989, Genes IV, John Wiley & Sons,
New York, N.Y.). For example, different strains of the same
microbial species may have a single or more nucleotide variation(s)
at the oligonucleotide hybridization site. The person skilled in
the art is well aware of the existence of variant algal, archaeal,
bacterial, fungal or parasitical DNA nucleic acids and/or sequences
for a specific gene and that the frequency of sequence variations
depends on the selective pressure during evolution on a given gene
product. The detection of a variant sequence for a region between
two PCR primers may be demonstrated by sequencing the amplification
product. In order to show the presence of sequence variants at the
primer hybridization site, one has to amplify a larger DNA target
with PCR primers outside that hybridization site. Sequencing of
this larger fragment will allow the detection of sequence variation
at this site. A similar strategy may be applied to show variants at
the hybridization site of a probe. Insofar as the divergence of the
target nucleic acids and/or sequences or a part thereof does not
affect the specificity and ubiquity of the amplification primers or
probes, variant microbial DNA is under the scope of this invention.
Variants of the selected primers or probes may also be used to
amplify or hybridize to a variant DNA.
Sequencing of tuf Nucleic Acids and/or Sequences from a Variety of
Archaeal, Bacterial, Fungal and Parasitical Species
[0125] The nucleotide sequence of a portion of tuf nucleic acids
and/or sequences was determined for a variety of archaeal,
bacterial, fungal and parasitical species. The amplification
primers (SEQ ID NOs. 664 and 697), which amplify a tuf gene portion
of approximately 890 bp, were used along with newly designed
sequencing primer pairs (See Annex I for the sequencing primers for
tuf nucleic acids and/or sequences). Most primer pairs can amplify
different copies of tuf genes (tufA and tufB). This is not
surprising since it is known that for several bacterial species
these two genes are nearly identical. For example, the entire tufA
and tufB genes from E. coli differ at only 13 nucleotide positions
(Neidhardt et al., 1996, Escherichia coli and Salmonella: Cellular
and Molecular Biology, 2.sup.nd ed., American Society for
Microbiology Press, Washington, D.C.). Similarly, some fungi are
known to have two nearly identical copies of tuf nucleic acids
and/or sequences (EF-1.quadrature.). These amplification primers
are degenerated at several nucleotide positions and contain
inosines in order to allow the amplification of a wide range of tuf
nucleic acids and/or sequences. The strategy used to select these
amplification primers is similar to that illustrated in Annex I for
the selection of universal primers. The tuf sequencing primers even
sometimes amplified highly divergent copies of tuf genes (tufC) as
illustrated in the case of some enterococcal species (SEQ ID NOs.:
73, 75, 76, 614 to 618, 621 and 987 to 989). To prove this, we have
determined the enterococcal tuf nucleic acids and/or sequences from
PCR amplicons cloned into a plasmid vector. Using the sequence data
from the cloned amplicons, we designed new sequencing primers
specific to the divergent (tufC) copy of enterococci (SEQ ID NOs.:
658-659 and 661) and then sequenced directly the tufC amplicons.
The amplification primers (SEQ ID NOs.: 543, 556, 557, 643-645,
660, 664, 694, 696 and 697) could be used to amplify the tuf
nucleic acids and/or sequences from any bacterial species. The
amplification primers (SEQ ID NOs.: 558, 559, 560, 653, 654, 655,
813, 815, 1974-1984, 1999-2003) could be used to amplify the tuf
(EF-1 .quadrature. genes from any fungal and/or parasitical
species. The amplification primers SEQ ID NOs. 1221-1228 could be
used to amplify bacterial tuf nucleic acids and/or sequences of the
EF-G subdivision (fusA) (FIG. 3). The amplification primers SEQ ID
NOs. 1224, and 1227-1229 could be used to amplify bacterial tuf
nucleic acids and/or sequences comprising the end of EF-G (fusA)
and the beginning of EF-Tu (tuf), including the intergenic region,
as shown in FIG. 3.
[0126] Most tuf fragments to be sequenced were amplified using the
following amplification protocol: One .mu.l of cell suspension (or
of purified genomic DNA 0.1-100 ng/.mu.l) was transferred directly
to 19 .mu.l of a PCR reaction mixture. Each PCR reaction contained
50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM
MgCl.sub.2, 1 .mu.M of each of the 2 primers, 200 .mu.M of each of
the four dNTPs, 0.5 unit of Taq DNA polymerase (Promega Corp.,
Madison, Wis.). PCR reactions were subjected to cycling using a
PTC-200 thermal cycler (MJ Research Inc., Watertown, Mass.) as
follows: 3 min at 94-96.degree. C. followed by 30-45 cycles of 1
min at 95.degree. C. for the denaturation step, 1 min at
50-55.degree. C. for the annealing step and 1 min at 72.degree. C.
for the extension step. Subsequently, twenty microliters of the
PCR-amplified mixture were resolved by electrophoresis in a 1.5%
agarose gel. The amplicons were then visualized by staining with
methylene blue (Flores et al., 1992, Biotechniques, 13:203-205).
The size of the amplification products was estimated by comparison
with a 100-bp molecular weight ladder. The band corresponding to
the specific amplification product was excised from the agarose gel
and purified using the QIAquick.TM. gel extraction kit (QIAGEN
Inc., Chatsworth, Calif.). The gel-purified DNA fragment was then
used directly in the sequencing protocol. Both strands of the tuf
genes amplification product were sequenced by the dideoxynucleotide
chain termination sequencing method by using an Applied Biosystems
automated DNA sequencer (model 377) with their Big Dye.TM.
Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems,
Foster City, Calif.). The sequencing reactions were performed by
using the same amplification primers and 10 ng/100 by of the
gel-purified amplicon per reaction. For the sequencing of long
amplicons such as those of eukaryotic tuf (EF-1 .quadrature.
nucleic acids and/or sequences, we designed internal sequencing
primers (SEQ ID NOs.: 654, 655 and 813) to be able to obtain
sequence data on both strands for most of the fragment length. In
order to ensure that the determined sequence did not contain errors
attributable to the sequencing of PCR artefacts, we have sequenced
two preparations of the gel-purified tuf amplification product
originating from two independent PCR amplifications. For most
target microbial species, the sequences determined for both
amplicon preparations were identical. In case of discrepancies,
amplicons from a third independent PCR amplification were
sequenced. Furthermore, the sequences of both strands were 100%
complementary thereby confirming the high accuracy of the
determined sequence. The tuf nucleic acids and/or sequences
determined using the above strategy are described in the Sequence
Listing. Table 7 gives the originating microbial species and the
source for each tuf sequence in the Sequence Listing.
[0127] The alignment of the tuf sequences determined by us or
selected from databases revealed clearly that the length of the
sequenced portion of the tuf genes is variable. There may be
insertions or deletions of several amino acids. In addition, in
several fungi introns were observed. Intron nucleic acids and/or
sequences are part of tuf nucleic acids and/or sequences and could
be useful in the design of species-specific primers and probes.
This explains why the size of the sequenced tuf amplification
products was variable from one fungal species to another.
Consequently, the nucleotide positions indicated on top of each of
Annexes IV to XX, XXIII to XXXI, XXXVIII and XLII do not correspond
for sequences having insertions or deletions.
[0128] It should also be noted that the various tuf nucleic acids
and/or sequences determined by us occasionally contain base
ambiguities. These degenerated nucleotides correspond to sequence
variations between tufA and tufB genes (or copies of the EF-G
subdivision of tuf nucleic acids and/or sequences, or copies of
EF-1 .quadrature. subdivision of tuf nucleic acids and/or sequences
for fungi and parasites) because the amplification primers amplify
both tuf genes. These nucleotide variations were not attributable
to nucleotide misincorporations by the Taq DNA polymerase because
the sequence of both strands was identical and also because the
sequences determined with both preparations of the gel-purified tuf
amplicons obtained from two independent PCR amplifications were
identical.
The Selection of Amplification Primers from Tuf Nucleic Acids
and/or Sequences
[0129] The tuf sequences determined by us or selected from public
databases were used to select PCR primers for universal detection
of bacteria, as well as for genus-specific, species-specific
family-specific or group-specific detection and identification. The
strategy used to select these PCR primers was based on the analysis
of multiple sequence alignments of various tuf sequences. For more
details about the selection of PCR primers from tuf sequences
please refer to Examples 5, 7-14, 17, 22, 24, 28, 30-31, 33, 36,
and 38-40, and to Annexes VI-IX, XI-XIX and XXV.
Sequencing of atpD and recA Nucleic Acids and/or Sequences from a
Variety of Archaeal, Bacterial, Fungal and Parasitical Species
[0130] The method used to obtain atpD and recA nucleic acids and/or
sequences is similar to that described above for tuf nucleic acids
and/or sequences.
The Selection of Amplification Primers from atpD or recA Nucleic
Acids and/or Sequences
[0131] The comparison of the nucleotide sequence for the atpD or
recA genes from various archaeal, bacterial, fungal and parasitical
species allowed the selection of PCR primers (refer to Examples 6,
13, 29, 34 and 37, and to Annexes IV, V, X, and XX).
DNA Amplification
[0132] For DNA amplification by the widely used PCR (polymerase
chain reaction) method, primer pairs were derived from proprietary
DNA fragments or from database sequences. Prior to synthesis, the
potential primer pairs were analyzed by using the Oligo.TM. 5.0
software to verify that they were good candidates for PCR
amplification.
[0133] During DNA amplification by PCR, two oligonucleotide primers
binding respectively to each strand of the heat-denatured target
DNA from the microbial genome are used to amplify exponentially in
vitro the target DNA by successive thermal cycles allowing
denaturation of the DNA, annealing of the primers and synthesis of
new targets at each cycle (Persing et al, 1993, Diagnostic
Molecular Microbiology: Principles and Applications, American
Society for Microbiology, Washington, D.C.).
[0134] Briefly, the PCR protocols were as follows: Treated clinical
specimens or standardized bacterial or fungal or parasitical
suspensions (see below) or purified genomic DNA from bacteria,
fungi or parasites were amplified in a 20 .mu.l PCR reaction
mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH
9.0), 2.5 mM MgCl.sub.2, 0.4 .mu.M of each primer, 200 .mu.M of
each of the four dNTPs and 0.5 unit of Taq DNA polymerase (Promega)
combined with the TaqStart.TM. antibody (Clontech Laboratories
Inc., Palo Alto, Calif.). The TaqStart.TM. antibody, which is a
neutralizing monoclonal antibody to Taq DNA polymerase, was added
to all PCR reactions to enhance the specificity and the sensitivity
of the amplifications (Kellogg et al., 1994, Biotechniques
16:1134-1137). The treatment of the clinical specimens varies with
the type of specimen tested, since the composition and the
sensitivity level required are different for each specimen type. It
consists in a rapid protocol to lyse the microbial cells and
eliminate or neutralize PCR inhibitors. For amplification from
bacterial or fungal or parasitical cultures or from purified
genomic DNA, the samples were added directly to the PCR
amplification mixture without any pre-treatment step. An internal
control was derived from sequences not found in the target
microorganisms or in the human genome. The internal control was
integrated into all amplification reactions to verify the
efficiency of the PCR assays and to ensure that significant PCR
inhibition was absent. Alternatively, an internal control derived
from rRNA was also useful to monitor the efficiency of microbial
lysis protocols.
[0135] PCR reactions were then subjected to thermal cycling (3 min
at 94-96.degree. C. followed by 30 cycles of 1 second at 95.degree.
C. for the denaturation step and 30 seconds at 50-65.degree. C. for
the annealing-extension step) using a PTC-200 thermal cycler (MJ
Research Inc.). The number of cycles performed for the PCR assays
varies according to the sensitivity level required. For example,
the sensitivity level required for microbial detection directly
from clinical specimens is higher for blood specimens than for
urine specimens because the concentration of microorganisms
associated with a septicemia can be much lower than that associated
with a urinary tract infection. Consequently, more sensitive PCR
assays having more thermal cycles are probably required for direct
detection from blood specimens. Similarly, PCR assays performed
directly from bacterial or fungal or parasitical cultures may be
less sensitive than PCR assays performed directly from clinical
specimens because the number of target organisms is normally much
lower in clinical specimens than in microbial cultures.
[0136] The person skilled in the art of DNA amplification knows the
existence of other rapid amplification procedures such as ligase
chain reaction (LCR), transcription-mediated amplification (TMA),
self-sustained sequence replication (3SR), nucleic acid
sequence-based amplification (NASBA), strand displacement
amplification (SDA), branched DNA (bDNA), cycling probe technology
(CPT), solid phase amplification (SPA), rolling circle
amplification technology (RCA), solid phase RCA, anchored SDA and
nuclease dependent signal amplification (NDSA) (Lee et al., 1997,
Nucleic Acid Amplification Technologies: Application to Disease
Diagnosis, Eaton Publishing, Boston, Mass.; Persing et al., 1993,
Diagnostic Molecular Microbiology: Principles and Applications,
American Society for Microbiology, Washington, D.C.; Westin et al.,
2000, Nat. Biotechnol. 18:199-204). The scope of this invention is
not limited to the use of amplification by PCR, but rather includes
the use of any rapid nucleic acid amplification method or any other
procedure which may be used to increase the sensitivity and/or the
rapidity of nucleic acid-based diagnostic tests. The scope of the
present invention also covers the use of any nucleic acids
amplification and detection technology including real-time or
post-amplification detection technologies, any amplification
technology combined with detection, any hybridization nucleic acid
chips or arrays technologies, any amplification chips or
combination of amplification and hybridization chips technologies.
Detection and identification by any sequencing method is also under
the scope of the present invention.
[0137] Any oligonucleotide suitable for the amplification of
nucleic acids by approaches other than PCR or for DNA hybridization
which are derived from the species-specific, genus-specific and
universal DNA fragments as well as from selected antimicrobial
agents resistance or toxin gene sequences included in this document
are also under the scope of this invention.
Detection of Amplification Products
[0138] Classically, detection of amplification is performed by
standard ethidium bromide-stained agarose gel electrophoresis. It
is clear that other methods for the detection of specific
amplification products, which may be faster and more practical for
routine diagnosis, may be used. Such methods may be based on the
detection of fluorescence after or during amplification. One simple
method for monitoring amplified DNA is to measure its rate of
formation by measuring the increase in fluorescence of
intercalating agents such as ethidium bromide or SYBR.RTM. Green I
(Molecular Probes). If more specific detection is required,
fluorescence-based technologies can monitor the appearance of a
specific product during the reaction. The use of dual-labeled
fluorogenic probes such as in the TaqMan.TM. system (Applied
Biosystems) which utilizes the 5'-3' exonuclease activity of the
Taq polymerase is a good example (Livak K. J. et al. 1995, PCR
Methods Appl. 4:357-362). TaqMan.TM. can be performed during
amplification and this "real-time" detection can be done in a
single closed tube hence eliminating post-PCR sample handling and
consequently preventing the risk of amplicon carryover. Several
other fluorescence-based detection methods can be performed in
real-time. Fluorescence resonance energy transfer (FRET) is the
principle behind the use of adjacent hybridization probes (Wittwer,
C. T. et al. 1997. BioTechniques 22:130-138), molecular beacons
(Tyagi S. and Kramer F. R. 1996. Nature Biotechnology 14:303-308)
and scorpions (Whitcomb et al. 1999. Nature Biotechnology
17:804-807). Adjacent hybridization probes are designed to be
internal to the amplification primers. The 3' end of one probe is
labelled with a donor fluorophore while the 5' end of an adjacent
probe is labelled with an acceptor fluorophore. When the two probes
are specifically hybridized in closed proximity (spaced by 1 to 5
nucleotides) the donor fluorophore which has been excited by an
external light source emits light that is absorbed by a second
acceptor that emit more fluorescence and yields a FRET signal.
Molecular beacons possess a stem-and-loop structure where the loop
is the probe and at the bottom of the stem a fluorescent moiety is
at one end while a quenching moiety is at the other end. The
beacons undergo a fluorogenic conformational change when they
hybridize to their targets hence separating the fluorochrome from
its quencher. The FRET principle is also used in an air thermal
cycler with a built-in fluorometer (Wittwer, C. T. et al. 1997.
BioTechniques 22:130-138). The amplification and detection are
extremely rapid as reactions are performed in capillaries: it takes
only 18 min to complete 45 cycles. Those techniques are suitable
especially in the case where few pathogens are searched for.
Boehringer-Roche Inc. sells the LightCycler.TM., and Cepheid makes
the SmartCycler. These two apparatus are capable of rapid cycle PCR
combined with fluorescent SYBR.RTM. Green I or FRET detection. We
recently demonstrated in our laboratory, real-time detection of 10
CFU in less than 40 minutes using adjacent hybridization probes on
the LightCycler.TM.. Methods based on the detection of fluorescence
are particularly promising for utilization in routine diagnosis as
they are very rapid, quantitative and can be automated.
[0139] Microbial pathogens detection and identification may also be
performed by solid support or liquid hybridization using
species-specific internal DNA probes hybridizing to an
amplification product. Such probes may be generated from any
sequence from our repertory and designed to specifically hybridize
to DNA amplification products which are objects of the present
invention. Alternatively, the internal probes for species or genus
or family or group detection and identification may be derived from
the amplicons produced by a universal, family-, group-, genus- or
species-specific amplification assay(s). The oligonucleotide probes
may be labeled with biotin or with digoxigenin or with any other
reporter molecule (for more details see below the section on hybrid
capture). Hybridization on a solid support is amendable to
miniaturization.
[0140] At present the oligonucleotide nucleic acid microarray
technology is appealing. Currently, available low to medium density
arrays (Heller et al., An integrated microelectronics hybridization
system for genomic research and diagnostic applications. In:
Harrison, D. J., and van den Berg, A., 1998, Micro total analysis
systems '98, Kluwer Academic Publisher, Dordrecht.) could
specifically capture fluorescent-labelled amplicons. Detection
methods for hybridization are not limited to fluorescence;
potentiometry, colorimetry and plasmon resonance are some examples
of alternative detection methods. In addition to detection by
hybridization, nucleic acid microarrays could be used to perform
rapid sequencing by hybridization. Mass spectrometry could also be
applicable for rapid identification of the amplicon or even for
sequencing of the amplification products (Chiu and Cantor, 1999,
Clinical Chemistry 45:1578; Berkenkamp et al., 1998, Science
281:260).
[0141] For the future of our assay format, we also consider the
major challenge of molecular diagnostics tools, i.e.: integration
of the major steps including sample preparation, genetic
amplification, detection, data analysis and presentation (Anderson
et al., Advances in integrated genetic analysis. In: Harrison, D.
J., and van den Berg, A., 1998, Micro total analysis systems '98,
Kluwer Academic Publisher, Dordrecht.).
[0142] To ensure PCR efficiency, glycerol, dimethyl sulfoxide
(DMSO) or other related solvents can be used to increase the
sensitivity of the PCR and to overcome problems associated with the
amplification of a target DNA having a high GC content or forming
strong secondary structures (Dieffenbach and Dveksler, 1995, PCR
Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Plainview, N.Y.). The concentration ranges for glycerol and DMSO
are 5-15% (v/v) and 3-10% (v/v), respectively. For the PCR reaction
mixture, the concentration ranges for the amplification primers and
MgCl.sub.2 are 0.1-1.5 .mu.M and 1.0-10.0 mM, respectively.
Modifications of the standard PCR protocol using external and
nested primers (i.e. nested PCR) or using more than one primer pair
(i.e. multiplex PCR) may also be used (Persing et al., 1993,
Diagnostic Molecular Microbiology: Principles and Applications,
American Society for Microbiology, Washington, D.C.). For more
details about the PCR protocols and amplicon detection methods, see
Examples.
Hybrid Capture and Chemiluminescence Detection of Amplification
Products
[0143] Hybridization and detection of amplicons by
chemiluminescence were adapted from Nikiforov et al. (1994, PCR
Methods and Applications 3:285-291 and 1995, Anal. Biochem.
227:201-209) and from the DIG.TM. system protocol of Boehringer
Mannheim. Briefly, 0.50 .mu.l of a 25 picomoles solution of capture
probe diluted in EDC {1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride) are immobilized in each well of
96-wells plates (Microlite.TM. 2, Dynex) by incubation overnight at
room temperature. The next day, the plates are incubated with a
solution of 1% BSA diluted into TNTw (10 mM Tris-HCl, pH 7.5; 150
mM NaCl; 0.05% Tween.TM.. 20) for 1 hour at 37.degree. C. The
plates are then washed on a Wellwash Ascent.TM. (Labsystems) with
TNTw followed by Washing Buffer (100 mM maleic acid pH7.5; 150 mM
NaCl; 0.3% Tween.TM. 20).
[0144] The amplicons were labelled with DIG-11-dUTP during PCR
using the PCR DIG Labelling Mix from Boehringer Mannheim according
to the manufacturer's instructions. Hybridization of the amplicons
to the capture probes is performed in triplicate at stringent
temperature (generally, probes are designed to allow hybridization
at 55.degree. C., the stringent temperature) for 30 minutes in 1.5
M NaCl; 10 mM EDTA. It is followed by two washes in 2.times.SSC;
0.1% SDS, then by four washes in 0.1.times.SSC; 0.1% SDS at the
stringent temperature (55.degree. C.). Detection with 1,2 dioxetane
chemiluminescent alkaline phosphatase substrates like CSPD.RTM.
(Tropix Inc.) is performed according to the manufacturer's
instructions but with shorter incubations times and a different
antibody concentration. The plates are agitated at each step, the
blocking incubation is performed for only 5 minutes, the
anti-DIG-AP1 is used at a 1:1000 dilution, the incubation with
antibody lasts 15 minutes, the plates are washed twice for only 5
minutes. Finally, after a 2 minutes incubation into the detection
buffer, the plates are incubated 5 minutes with CSPD.RTM. at room
temperature followed by a 10 minutes incubation at 37.degree. C.
without agitation. Luminous signal detection is performed on a
Dynex Microtiter Plate Luminometer using RLU (Relative Light
Units).
Specificity, Ubiquity and Sensitivity Tests for Oligonucleotide
Primers and Probes
[0145] The specificity of oligonucleotide primers and probes was
tested by amplification of DNA or by hybridization with bacterial
or fungal or parasitical species selected from a panel comprising
closely related species and species sharing the same
anatomo-pathological site (see Annexes and Examples). All of the
bacterial, fungal and parasitical species tested were likely to be
pathogens associated with infections or potential contaminants
which can be isolated from clinical specimens. Each target DNA
could be released from microbial cells using standard chemical
and/or physical treatments to lyse the cells (Sambrook et al.,
1989, Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) or
alternatively, genomic DNA purified with the GNOME.TM. DNA kit
(Bio101, Vista, Calif.) was used. Subsequently, the DNA was
subjected to amplification with the primer pairs. Specific primers
or probes amplified only the target microbial species, genus,
family or group.
[0146] Oligonucleotides primers found to amplify specifically the
target species, genus, family or group were subsequently tested for
their ubiquity by amplification (i.e. ubiquitous primers amplified
efficiently most or all isolates of the target species or genus or
family or group). Finally, the sensitivity of the primers or probes
was determined by using 10-fold or 2-fold dilutions of purified
genomic DNA from the targeted microorganism. For most assays,
sensitivity levels in the range of 1-100 copies were obtained. The
specificity, ubiquity and sensitivity of the PCR assays using the
selected amplification primer pairs were tested either directly
from cultures of microbial species or from purified microbial
genomic DNA.
[0147] Probes were tested in hybrid capture assays as described
above. An oligonucleotide probe was considered specific only when
it hybridized solely to DNA from the species or genus or family or
group from which it was selected. Oligonucleotide probes found to
be specific were subsequently tested for their ubiquity (i.e.
ubiquitous probes detected efficiently most or all isolates of the
target species or genus or family or group) by hybridization to
microbial DNAs from different clinical isolates of the species or
genus or family or group of interest including ATCC reference
strains. Similarly, oligonucleotide primers and probes could be
derived from antimicrobial agents resistance or toxin genes which
are objects of the present invention.
Reference Strains
[0148] The reference strains used to build proprietary tuf, atpD
and recA sequence data subrepertories, as well as to test the
amplification and hybridization assays were obtained from (i) the
American Type Culture Collection (ATCC), (ii) the Laboratoire de
sante publique du Quebec (LSPQ), (iii) the Centers for Disease
Control and Prevention (CDC), (iv) the National Culture Type
Collection (NCTC) and (v) several other reference laboratories
throughout the world. The identity of our reference strains was
confirmed by phenotypic testing and reconfirmed by analysis of tuf,
atpD and recA sequences (see Example 13).
Antimicrobial Agents Resistance Genes
[0149] Antimicrobial resistance complicates treatment and often
leads to therapeutic failures. Furthermore, overuse of antibiotics
inevitably leads to the emergence of microbial resistance. Our goal
is to provide clinicians, in approximately one hour, the needed
information to prescribe optimal treatments. Besides the rapid
identification of negative clinical specimens with DNA-based tests
for universal algal, archaeal, bacterial, fungal or parasitical
detection and the identification of the presence of a specific
pathogen in the positive specimens with species- and/or genus-
and/or family- and/or group-specific DNA-based tests, clinicians
also need timely information about the ability of the microbial
pathogen to resist antibiotic treatments. We feel that the most
efficient strategy to evaluate rapidly microbial resistance to
antimicrobials is to detect directly from the clinical specimens
the most common and clinically important antimicrobial agents
resistance genes (i.e. DNA-based tests for the specific detection
of antimicrobial agents resistance genes). Since the sequence from
the most important and common antimicrobial agents resistance genes
are available from public databases, our strategy is to use the
sequence from a portion or from the entire resistance gene to
design specific oligonucleotide primers or probes which will be
used as a basis for the development of sensitive and rapid
DNA-based tests. The list of each of the antimicrobial agents
resistance genes selected on the basis of their clinical relevance
(i.e. high incidence and importance) is given in Table 5;
descriptions of the designed amplification primers and internal
probes are given in Annexes XXXIV-XXXVII, XXXIX, XLV, and L-LI. Our
approach is unique because the antimicrobial agents resistance
genes detection and the microbial detection and identification can
be performed simultaneously, or independently, or sequentially in
multiplex or parallel or sequential assays under uniform PCR
amplification conditions. These amplifications can also be done
separately.
Toxin Genes
[0150] Toxin identification is often very important to prescribe
optimal treatments. Besides the rapid identification of negative
clinical specimens with DNA-based tests for universal bacterial
detection and the identification of the presence of a specific
pathogen in the positive specimens with species- and/or genus-
and/or family- and/or group-specific DNA-based tests, clinicians
sometimes need timely information about the ability of certain
bacterial pathogens to produce toxins. Since the sequence from the
most important and common bacterial toxin genes are available from
public databases, our strategy is to use the sequence from a
portion or from the entire toxin gene to design specific
oligonucleotide primers or probes which will be used as a basis for
the development of sensitive and rapid DNA-based tests. The list of
each of the bacterial toxin genes selected on the basis of their
clinical relevance (i.e. high incidence and importance) is given in
Table 6; descriptions of the designed amplification primers and
internal probes are given in Annexes XXII, XXXII and XXXIII. Our
approach is unique because the toxin genes detection and the
bacterial detection and identification can be performed
simultaneously, or independently, or sequentially, in multiplex or
parallel or sequential assays under uniform PCR amplification
conditions. These amplifications can also be done separately.
Universal Bacterial Detection
[0151] In the routine microbiology laboratory, a high percentage of
clinical specimens sent for bacterial identification are negative
by culture. Testing clinical samples with universal amplification
primers or universal probes to detect the presence of bacteria
prior to specific identification and screening out the numerous
negative specimens is thus useful as it reduces costs and may
rapidly orient the clinical management of the patients. Several
amplification primers and probes were therefore synthesized from
highly conserved portions of bacterial sequences from the tuf, atpD
and recA nucleic acids and/or sequences. The universal primers
selection was based on a multiple sequence alignment constructed
with sequences from our repertory.
[0152] All computer analysis of amino acid and nucleotide sequences
were performed by using the GCG programs. Subsequently, optimal PCR
primers for the universal amplification of bacteria were selected
with the help of the Oligo.TM. program. The selected primers are
degenerated at several nucleotide positions and contain several
inosines in order to allow the amplification of all clinically
relevant bacterial species. Inosine is a nucleotide analog able to
specifically bind to any of the four nucleotides A, C, G or T.
Degenerated oligonucleotides consist of an oligonucleotide mix
having two or more of the four nucleotides A, C, G or T at the site
of mismatches. The inclusion of inosine and/or of base ambiguities
in the amplification primers allow mismatch tolerance thereby
permitting the amplification of a wider array of target nucleotide
sequences (Dieffenbach and Dveksler, 1995 PCR Primer: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
[0153] The amplification conditions with the universal primers are
very similar to those used for the species- and genus-specific
amplification assays except that the annealing temperature is
slightly lower. The original universal PCR assay described in our
assigned WO98/20157 (SEQ ID NOs. 23-24 of the latter application)
was specific and nearly ubiquitous for the detection of bacteria.
The specificity for bacteria was verified by amplifying genomic DNA
isolated from the 12 fungal species as well as genomic DNA from
Leishmania donovani, Saccharomyces cerevisiae and human
lymphocytes. None of the above eukaryotic DNA preparations could be
amplified by the universal assay, thereby suggesting that this test
is specific for bacteria. The ubiquity of the universal assay was
verified by amplifying genomic DNAs from 116 reference strains
which represent 95 of the most clinically relevant bacterial
species. These species have been selected from the bacterial
species listed in Table 4. We found that at least 104 of these
strains could be amplified. However, the assay could be improved
since bacterial species which could not be amplified with the
original tuf nucleic acids and/or sequences-based assay included
species belonging to the following genera: Corynebacterium (11
species) and Stenotrophomonas (1 species). Sequencing of the tuf
genes from these bacterial species and others has been performed in
the scope of the present invention in order to improve the
universal assay. This sequencing data has been used to select new
universal primers which may be more ubiquitous and more sensitive.
Also, we improved our primer and probes design strategy by taking
into consideration the phylogeny observed in analysing our
repertory of tuf, atpD and recA sequences. Data from each of the 3
main subrepertories (tuf, atpD and recA) was subjected to a basic
phylogenic analysis using the Pileup command from version 10 of the
GCG package (Genetics Computer Group, inc.). This analysis
indicated the main branches or phyla reflecting the relationships
between sequences. Instead of trying to design primers or probes
able to hybridize to all phyla, we designed primers or probes able
to hybridize to the main phyla while trying to use the largest
phylum possible. This strategy should allow less degenerated
primers hence improving sensitivity and by combining primers in a
multiplex assay, improve ubiquity. Universal primers SEQ ID NOs.
643-645 based on tuf sequences have been designed to amplify most
pathogenic bacteria except Actinomyceteae, Clostridiaceae and the
Cytophaga, Flexibacter and Bacteroides phylum (pathogenic bacteria
of this phylum include mostly Bacteroides, Porphyromonas and
Prevotella species). Primers to fill these gaps have been designed
for Actinomyceteae (SEQ ID NOs. 646-648), Clostridiaceae (SEQ ID
NOs. 796-797, 808-811), and the Cytophaga, Flexibacter and
Bacteroides phylum (SEQ ID NOs. 649-651), also derived from tuf
nucleic acids and/or sequences. These primers sets could be used
alone or in conjunction to render the universal assay more
ubiquitous.
[0154] Universal primers derived from atpD sequences include SEQ ID
NOs. 562-565. Combination of these primers does not amplify human
DNA but should amplify almost all pathogenic bacterial species
except proteobacteria belonging to the epsilon subdivision
(Campylobacter and Helicobacter), the bacteria from the Cytophaga,
Flexibacter and Bacteroides group and some actinomycetes and
corynebacteria. By analysing atpD sequences from the latter
species, primers and probes to specifically fill these gaps could
be designed and used in conjunction with primers SEQ ID NOs.
562-565, also derived from atpD nucleic acids and/or sequences.
[0155] In addition, universality of the assay could be expanded by
mixing atpD sequences-derived primers with tuf sequences-derived
primers. Ultimately, even recA sequences-derived primers could be
added to fill some gaps in the universal assay.
[0156] It is important to note that the 95 bacterial species
selected to test the ubiquity of the universal assay include all of
the most clinically relevant bacterial species associated with a
variety of human infections acquired in the community or in
hospitals (nosocomial infections). The most clinically important
bacterial and fungal pathogens are listed in Tables 1 and 2.
Amino Acid Sequences Derived from tuf, atpD and recA Nucleic Acids
and/or Sequences
[0157] The amino acid sequences translated from the repertory of
tuf, atpD and recA nucleic acids and/or sequences are also an
object of the present invention. The amino acid sequence data will
be particularly useful for homology modeling of three-dimensional
(3D) structure of the elongation factor Tu, elongation factor G,
elongation factor 1.alpha., ATPase subunit beta and RecA
recombinase. For all these proteins, at least one structure model
has been published using X-ray diffraction data from crystals.
Based on those structural informations it is possible to use
computer software to build 3D model structures for any other
protein having peptide sequence homologies with the known structure
(Greer, 1991, Methods in Enzymology, 202:239-252; Taylor, 1994,
Trends Biotechnol., 12(5):154-158; Sali, 1995, Curr. Opin.
Biotechnol. 6:437-451; Sanchez and Sali, 1997, Curr. Opin. Struct.
Biol. 7:206-214; Fischer and Eisenberg, 1999, Curr. Opin. Struct.
Biol. 9:208-211; Guex et al., 1999, Trends Biochem. Sci. 24:
364-367). Model structures of target proteins are used for the
design or to predict the behavior of ligands and inhibitors such as
antibiotics. Since EF-Tu and EF-G are already known as antibiotic
targets (see above) and since the beta subunit of ATPase and RecA
recombinase are essential to the survival of the microbial cells in
natural conditions of infection, all four proteins could be
considered antibiotic targets. Sequence data, especially the new
data generated by us could be very useful to assist the creation of
new antibiotic molecules with desired spectrum of activity. In
addition, model structures could be used to improve protein
function for commercial purposes such as improving antibiotic
production by microbial strains or increasing biomass.
[0158] The following detailed embodiments and appended drawings are
provided as illustrative examples of his invention, with no
intention to limit the scope thereof.
EXAMPLES AND ANNEXES
[0159] For sake of clarity, here is a list of Examples and
Annexes:
[0160] Example 1: Sequencing of bacterial atpD (F-type and V-type)
gene fragments.
[0161] Example 2: Sequencing of eukaryotic atpD (F-type and V-type)
gene fragments.
[0162] Example 3: Sequencing of eukaryotic tuf (EF-1) gene
fragments.
[0163] Example 4: Sequencing of eukaryotic tuf (organelle origin,
M) gene fragments.
[0164] Example 5: Specific detection and identification of
Streptococcus agalactiae using tuf sequences.
[0165] Example 6: Specific detection and identification of
Streptococcus agalactiae using atpD sequences.
[0166] Example 7: Development of a PCR assay for detection and
identification of staphylococci at genus and species levels.
[0167] Example 8: Differentiating between the two closely related
yeast species Candida albicans and Candida dubliniensis.
[0168] Example 9: Specific detection and identification of
Entamoeba histolytica.
[0169] Example 10: Sensitive detection and identification of
Chlamydia trachomatis.
[0170] Example 11: Genus-specific detection and identification of
enterococci.
[0171] Example 12: Detection and identification of the major
bacterial platelets contaminants using tuf sequences with a
multiplex PCR test.
[0172] Example 13: The resolving power of the tuf and atpD
sequences databases is comparable to the biochemical methods for
bacterial identification.
[0173] Example 14: Detection of group B streptococci from clinical
specimens.
[0174] Example 15: Simultaneous detection and identification of
Streptococcus pyogenes and its pyrogenic exotoxin A.
[0175] Example 16: Real-time detection and identification of Shiga
toxin-producing bacteria.
[0176] Example 17: Development of a PCR assay for the detection and
identification of staphylococci at genus and species levels and its
associated mecA gene.
[0177] Example 18: Sequencing of pbp1a, pbp2b and pbp2x genes of
Streptococcus pneumoniae.
[0178] Example 19: Sequencing of hexA genes of Streptococcus
species.
[0179] Example 20: Development of a multiplex PCR assay for the
detection of Streptococcus pneumoniae and its penicillin resistance
genes.
[0180] Example 21: Sequencing of the vancomycin resistance vanA,
vanC1, vanC2 and vanC3 genes.
[0181] Example 22: Development of a PCR assay for the detection and
identification of enterococci at genus and species levels and its
associated resistance genes vanA and vanB.
[0182] Example 23: Development of a multiplex PCR assay for
detection and identification of vancomycin-resistant Enterococcus
faecalis, Enterococcus faecium, Enterococcus gallinarum,
Enterococcus casseliflavus, and Enterococcus flavescens.
[0183] Example 24: Universal amplification involving the EF-G
(fusA) subdivision of tuf sequences.
[0184] Example 25: DNA fragment isolation from Staphylococcus
saprophyticus by arbitrarily primed PCR.
[0185] Example 26: Sequencing of prokaryotic tuf gene
fragments.
[0186] Example 27: Sequencing of procaryotic recA gene
fragments.
[0187] Example 28: Specific detection and identification of
Escherichia coli/Shigella sp. using tuf sequences.
[0188] Example 29: Specific detection and identification of
Klebsiella pneumoniae using atpD sequences.
[0189] Example 30: Specific detection and identification of
Acinetobacter baumanii using tuf sequences.
[0190] Example 31: Specific detection and identification of
Neisseria gonorrhoeae using tuf sequences.
[0191] Example 32: Sequencing of bacterial gyrA and parC gene
fragments.
[0192] Example 33: Development of a PCR assay for the specific
detection and identification of Staphylococcus aureus and its
quinolone resistance genes gyrA and parC.
[0193] Example 34: Development of a PCR assay for the detection and
identification of Klebsiella pneumoniae and its quinolone
resistance genes gyrA and parC.
[0194] Example 35: Development of a PCR assay for the detection and
identification of Streptococcus pneumoniae and its quinolone
resistance genes gyrA and parC.
[0195] Example 36: Detection of extended-spectrum TEM-type
.beta.-lactamases in Escherichia coli.
[0196] Example 37: Detection of extended-spectrum SHV-type
.beta.-lactamases in Klebsiella pneumoniae.
[0197] Example 38: Development of a PCR assay for the detection and
identification of Neisseria gonorrhoeae and its associated
tetracycline resistance gene tetM.
[0198] Example 39: Development of a PCR assay for the detection and
identification of Shigella sp. and their associated trimethoprim
resistance gene dhfrIa.
[0199] Example 40: Development of a PCR assay for the detection and
identification of Acinetobacter baumanii and its associated
aminoglycoside resistance gene aph(3')-VIa.
[0200] Example 41: Specific detection and identification of
Bacteroides fragilis using atpD (V-type) sequences.
[0201] Example 42: Evidence for horizontal gene transfer in the
evolution of the elongation factor Tu in Enterococci.
[0202] Example 43: Elongation factor Tu (tuf) and the F-ATPase
beta-subunit (atpD) as phylogenetic tools for species of the family
Enterobacteriaceae.
[0203] Example 44: Testing new pairs of PCR primers selected from
two species-specific genomic DNA fragments which are objects of
U.S. Pat. No. 6,001,564.
[0204] Example 45: Testing modified versions of PCR primers derived
from the sequence of several primers which are objects of U.S. Pat.
No. 6,001,564.
[0205] The various Annexes show the strategies used for the
selection of a variety of DNA amplification primers, nucleic acid
hybridization probes and molecular beacon internal probes: [0206]
(i) Annex I shows the amplification primers used for nucleic acid
amplification from tuf sequences. [0207] (ii) Annex II shows the
amplification primers used for nucleic acid amplification from atpD
sequences. [0208] (iii) Annex III shows the internal hybridization
probes for detection of tuf sequences. [0209] (iv) Annex IV
illustrates the strategy used for the selection of the
amplification primers specific for atpD sequences of the F-type.
[0210] (v) Annex V illustrates the strategy used for the selection
of the amplification primers specific for atpD sequences of the
V-type. [0211] (vi) Annex VI illustrates the strategy used for the
selection of the amplification primers specific for the tuf
sequences of organelle lineage (M, the letter M is used to indicate
that in most cases, the organelle is the mitochondria). [0212]
(vii) Annex VII illustrates the strategy used for the selection of
the amplification primers specific for the tuf sequences of
eukaryotes (EF-1). [0213] (viii) Annex VIII illustrates the
strategy for the selection of Streptococcus agalactiae-specific
amplification primers from tuf sequences. [0214] (ix) Annex IX
illustrates the strategy for the selection of Streptococcus
agalactiae-specific hybridization probes from tuf sequences. [0215]
(x) Annex X illustrates the strategy for the selection of
Streptococcus agalactiae-specific amplification primers from atpD
sequences. [0216] (xi) Annex XI illustrates the strategy for the
selection from tuf sequences of Candida
albicans/dubliniensis-specific amplification primers, Candida
albicans-specific hybridization probe and Candida
dubliniensis-specific hybridization probe. [0217] (xii) Annex XII
illustrates the strategy for the selection of
Staphylococcus-specific amplification primers from tuf sequences.
[0218] (xiii) Annex XIII illustrates the strategy for the selection
of the Staphylococcus-specific hybridization probe from tuf
sequences. [0219] (xiv) Annex XIV illustrates the strategy for the
selection of Staphylococcus saprophyticus-specific and
Staphylococcus haemolyticus-specific hybridization probes from tuf
sequences. [0220] (xv) Annex XV illustrates the strategy for the
selection of Staphylococcus aureus-specific and Staphylococcus
epidermidis-specific hybridization probes from tuf sequences.
[0221] (xvi) Annex XVI illustrates the strategy for the selection
of the Staphylococcus hominis-specific hybridization probe from tuf
sequences. [0222] (xvii) Annex XVII illustrates the strategy for
the selection of the Enterococcus-specific amplification primers
from tuf sequences. [0223] (xviii) Annex XVIII illustrates the
strategy for the selection of the Enterococcus faecalis-specific
hybridization probe, of the Enterococcus faecium-specific
hybridization probe and of the Enterococcus
casseliflavus-flavescens-gallinarum group-specific hybridization
probe from tuf sequences. [0224] (xix) Annex XIX illustrates the
strategy for the selection of primers from tuf sequences for the
identification of platelets contaminants. [0225] (xx) Annex XX
illustrates the strategy for the selection of the universal
amplification primers from atpD sequences. [0226] (xxi) Annex XXI
shows the amplification primers used for nucleic acid amplification
from recA sequences. [0227] (xxii) Annex XXII shows the specific
and ubiquitous primers for nucleic acid amplification from speA
sequences. [0228] (xxiii) Annex XXIII illustrates the first
strategy for the selection of Streptococcus pyogenes-specific
amplification primers from speA sequences. [0229] (xxiv) Annex XXIV
illustrates the second strategy for the selection of Streptococcus
pyogenes-specific amplification primers from speA sequences. [0230]
(xxv) Annex XXV illustrates the strategy for the selection of
Streptococcus pyogenes-specific amplification primers from tuf
sequences. [0231] (xxvi) Annex XXVI illustrates the strategy for
the selection of stx.sub.1-specific amplification primers and
hybridization probe. [0232] (xxvii) Annex XXVII illustrates the
strategy for the selection of stx.sub.2-specific amplification
primers and hybridization probe. [0233] (xxviii) Annex XXVIII
illustrates the strategy for the selection of vanA-specific
amplification primers from van sequences. [0234] (xxix) Annex XXIX
illustrates the strategy for the selection of vanB-specific
amplification primers from van sequences. [0235] (xxx) Annex XXX
illustrates the strategy for the selection of vanC-specific
amplification primers from vanC sequences. [0236] (xxxi) Annex XXXI
illustrates the strategy for the selection of Streptococcus
pneumoniae-specific amplification primers and hybridization probes
from pbp1a sequences. [0237] (xxxii) Annex XXXII shows the specific
and ubiquitous primers for nucleic acid amplification from toxin
gene sequences. [0238] (xxxiii) Annex XXXIII shows the molecular
beacon internal hybridization probes for specific detection of
toxin sequences. [0239] (xxxiv) Annex XXXIV shows the specific and
ubiquitous primers for nucleic acid amplification from van
sequences. [0240] (xxxv) Annex XXXV shows the internal
hybridization probes for specific detection of van sequences.
[0241] (xxxvi) Annex XXXVI shows the specific and ubiquitous
primers for nucleic acid amplification from pbp sequences. [0242]
(xxxvii) Annex XXXVII shows the internal hybridization probes for
specific detection of pbp sequences. [0243] (xxxviii) Annex XXXVIII
illustrates the strategy for the selection of vanAB-specific
amplification primers and vanA- and vanB-specific hybridization
probes from van sequences. [0244] (xxxix) Annex XXXIX shows the
internal hybridization probe for specific detection of mecA. [0245]
(xl) Annex XL shows the specific and ubiquitous primers for nucleic
acid amplification from hexA sequences. [0246] (xli) Annex XLI
shows the internal hybridization probe for specific detection of
hexA. [0247] (xlii) Annex XLII illustrates the strategy for the
selection of Streptococcus pneumoniae species-specific
amplification primers and hybridization probe from hexA sequences.
[0248] (xliii) Annex XLIII shows the specific and ubiquitous
primers for nucleic acid amplification from pcp sequences. [0249]
(xliv) Annex XLIV shows specific and ubiquitous primers for nucleic
acid amplification of S. saprophyticus sequences of unknown coding
potential. [0250] (xlv) Annex XLV shows the molecular beacon
internal hybridization probes for specific detection of
antimicrobial agents resistance gene sequences. [0251] (xlvi) Annex
XLVI shows the molecular beacon internal hybridization probe for
specific detection of S. aureus gene sequences of unknown coding
potential. [0252] (xlvii) Annex XLVII shows the molecular beacon
hybridization internal probe for specific detection of tuf
sequences. [0253] (xlviii) Annex XLVIII shows the molecular beacon
internal hybridization probes for specific detection of ddI and mtl
sequences. [0254] (xlix) Annex XLIX shows the internal
hybridization probe for specific detection of S. aureus sequences
of unknown coding potential. [0255] (l) Annex L shows the
amplification primers used for nucleic acid amplification from
antimicrobial agents resistance genes sequences. [0256] (li) Annex
LI shows the internal hybridization probes for specific detection
of antimicrobial agents resistance genes sequences. [0257] (lii)
Annex LII shows the molecular beacon internal hybridization probes
for specific detection of atpD sequences. [0258] (liii) Annex LIII
shows the internal hybridization probes for specific detection of
atpD sequences. [0259] (liv) Annex LIVI shows the internal
hybridization probes for specific detection of ddI and mtl
sequences.
[0260] As shown in these Annexes, the selected amplification
primers may contain inosines and/or base ambiguities. Inosine is a
nucleotide analog able to specifically bind to any of the four
nucleotides A, C, G or T. Alternatively, degenerated
oligonucleotides which consist of an oligonucleotide mix having two
or more of the four nucleotides A, C, G or T at the site of
mismatches were used. The inclusion of inosine and/or of
degeneracies in the amplification primers allows mismatch tolerance
thereby permitting the amplification of a wider array of target
nucleotide sequences (Dieffenbach and Dveksler, 1995 PCR Primer: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview,
N.Y.).
EXAMPLES
Example 1
Sequencing of Bacterial atpD (F-Type and V-Type) Gene Fragments
[0261] As shown in Annex IV, the comparison of publicly available
atpD (F-type) sequences from a variety of bacterial species
revealed conserved regions allowing the design of PCR primers able
to amplify atpD sequences (F-type) from a wide range of bacterial
species. Using primers pairs SEQ ID NOs. 566 and 567, 566 and 814,
568 and 567, 570 and 567, 572 and 567, 569 and 567, 571 and 567,
700 and 567, it was possible to amplify and sequence atpD sequences
SEQ ID NOs. 242-270, 272-398, 673-674, 737-767, 866-867, 942-955,
1245-1254, 1256-1265, 1527, 1576, 1577, 1600-1604, 1640-1646, 1649,
1652, 1655, 1657, 1659-1660, 1671, 1844-1845, and 1849-1865.
[0262] Similarly, Annex V shows the strategy to design the PCR
primers able to amplify atpD sequences of the V-type from a wide
range of archaeal and bacterial species. Using primers SEQ ID NOs.
681-683, it was possible to amplify and sequence atpD sequences SEQ
ID NOs. 827-832, 929-931, 958 and 966. As the gene was difficult to
amplify for several species, additional amplification primers were
designed inside the original amplicon (SEQ ID NOs. 1203-1207) in
order to obtain sequence information for these species. Other
primers (SEQ ID NO. 1212, 1213, 2282-2285) were also designed to
amplify regions of the atpD gene (V-type) in archaebacteria.
Example 2
Sequencing of Eukaryotic atpD (F-Type and V-Type) Gene
Fragments
[0263] The comparison of publicly available atpD (F-type) sequences
from a variety of fungal and parasitical species revealed conserved
regions allowing the design of PCR primers able to amplify atpD
sequences from a wide range of fungal and parasitical species.
Using primers pairs SEQ ID NOs. 568 and 573, 574 and 573, 574 and
708, and 566 and 567, it was possible to amplify and sequence atpD
sequences SEQ ID NOs. 458-497, 530-538, 663, 667, 676, 678-680,
768-778, 856-862, 889-896, 941, 1638-1639, 1647, 1650-1651,
1653-1654, 1656, 1658, 1684, 1846-1848, and 2189-2192.
[0264] In the same manner, the primers described in Annex V (SEQ ID
NOs. 681-683) could amplify the atpD (V-type) gene from various
fungal and parasitical species. This strategy allowed to obtain SEQ
ID NOs. 834-839, 956-957, and 959-965.
Example 3
Sequencing of Eukaryotic tuf (EF-1) Gene Fragments
[0265] As shown in Annex VII, the comparison of publicly available
tuf (EF-1) sequences from a variety of fungal and parasitical
species revealed conserved regions allowing the design of PCR
primers able to amplify tuf sequences from a wide range of fungal
and parasitical species. Using primers pairs SEQ ID NOs. 558 and
559, 813 and 559, 558 and 815, 560 and 559, 653 and 559, 558 and
655, and 654 and 559, 1999 and 2000, 2001 and 2003, 2002 and 2003,
it was possible to amplify and sequence tuf sequences SEQ ID NOs.
399-457, 509-529, 622-624, 677, 779-790, 840-842, 865, 897-903,
1266-1287, 1561-1571 and 1685.
Example 4
Sequencing of Eukaryotic tuf (Organelle Origin, M) Gene
Fragments
[0266] As shown in Annex VI, the comparison of publicly available
tuf (organelle origin, M) sequences from a variety of fungal and
parasitical organelles revealed conserved regions allowing the
design of PCR primers able to amplify tuf sequences of several
organelles belonging to a wide range fungal and parasitical
species. Using primers pairs SEQ ID NOs. 664 and 652, 664 and 561,
911 and 914, 912 and 914, 913 and 915, 916 and 561, 664 and 917, it
was possible to amplify and sequence tuf sequences SEQ ID NOs.
498-508, 791-792, 843-855, 904-910, 1664, 1666-1667, 1669-1670,
1673-1683, 1686-1689, 1874-1876, 1879, 1956-1960, and
2193-2199.
Example 5
Specific Detection and Identification of Streptococcus agalactiae
Using tuf Sequences
[0267] As shown in Annex VIII, the comparison of tuf sequences from
a variety of bacterial species allowed the selection of PCR primers
specific for S. agalactiae. The strategy used to design the PCR
primers was based on the analysis of a multiple sequence alignment
of various tuf sequences. The multiple sequence alignment includes
the tuf sequences of four bacterial strains from the target species
as well as tuf sequences from other species and bacterial genera,
especially representatives of closely related species. A careful
analysis of this alignment allowed the selection of oligonucleotide
sequences which are conserved within the target species but which
discriminate sequences from other species and genera, especially
from the closely related species, thereby permitting the
species-specific, ubiquitous and sensitive detection and
identification of the target bacterial species.
[0268] The chosen primer pair, oligos SEQ ID NO. 549 and SEQ ID NO.
550, gives an amplification product of 252 bp. Standard PCR was
carried out using 0.4 .mu.M of each primer, 2.5 mM MgCl.sub.2, BSA
0.05 mM, 1.times. Taq Buffer (Promega), dNTP 0.2 mM (Pharmacia),
0.5 U Taq DNA polymerase (Promega) coupled with TaqStart.TM.
antibody (Clontech Laboratories Inc., Palo Alto), 1 .mu.l of
genomic DNA sample in a final volume of 20 .mu.l using a PTC-200
thermocycler (MJ Research Inc.). The optimal cycling conditions for
maximum sensitivity and specificity were 3 minutes at 95.degree. C.
for initial denaturation, then forty cycles of two steps consisting
of 1 second at 95.degree. C. and 30 seconds at 62.degree. C.,
followed by terminal extension at 72.degree. C. for 2 minutes.
Detection of the PCR products was made by electrophoresis in
agarose gels (2%) containing 0.25 .mu.g/ml of ethidium bromide.
[0269] Specificity of the assay was tested by adding into the PCR
reactions, 0.1 ng of genomic DNA from each of the bacterial species
listed in Table 8. Efficient amplification was observed only for
the 5 S. agalactiae strains listed. Of the other bacterial species,
including 32 species representative of the vaginal flora and 27
other streptococcal species, only S. acidominimus yielded
amplification. The signal with 0.1 ng of S. acidominimus genomic
DNA was weak and the detection limit for this species was 10 pg
(corresponding to more than 4000 genome copies) while the detection
limit for S. agalactiae was 2.5 fg (corresponding to one genome
copy) of genomic DNA.
[0270] To increase the specificity of the assay, internal probes
were designed for FRET (Fluorescence Resonance Energy Transfer)
detection using the LightCycler.TM. (Idaho Technology). As
illustrated in Annex IX, a multiple sequence alignment of
streptococcal tuf sequence fragments corresponding to the 252 by
region amplified by primers SEQ ID NO. 549 and SEQ ID NO. 550, was
used for the design of internal probes TSagHF436 (SEQ ID NO. 582)
and TSagHF465 (SEQ ID NO. 583). The region of the amplicon selected
for internal probes contained sequences unique and specific to S.
agalactiae. SEQ ID NO. 583, the more specific probe, is labelled
with fluorescein in 3', while SEQ ID NO. 582, the less discriminant
probe, is labelled with CY5 in 5' and blocked in 3' with a
phosphate group. However, since the FRET signal is only emitted if
both probes are adjacently hybridized on the same target amplicon,
detection is highly specific.
[0271] Real-time detection of PCR products using the
LightCycler.TM. was carried out using 0.4 .mu.M of each primer (SEQ
ID NO. 549-550), 0.2 .mu.M of each probe (SEQ ID NO. 582-583), 2.5
mM MgCl.sub.2, BSA 450 .mu.g/ml, 1.times.PC2 Buffer (AB Peptides,
St-Louis, dNTP 0.2 mM (Pharmacia), 0.5 U KlenTaq1.TM. DNA
polymerase (AB Peptides) coupled with TaqStart.TM. antibody
(Clontech Laboratories Inc., Palo Alto), 0.7 .mu.l of genomic DNA
sample in a final volume of 7 .mu.l using a LightCycler
thermocycler (Idaho Technology). The optimal cycling conditions for
maximum sensitivity and specificity were 3 minutes at 94.degree. C.
for initial denaturation, then forty cycles of three steps
consisting of 0 second (this setting meaning the LightCycler will
reach the target temperature and stay at it for its minimal amount
of time) at 94.degree. C., 10 seconds at 64.degree. C., 20 seconds
at 72.degree. C. Amplification was monitored during each annealing
steps using the fluorescence ratio. The streptococcal species
having close sequence homologies with the tuf sequence of S.
agalactiae (S. acidominimus, S. anginosus, S. bovis, S.
dysgalactiae, S. equi, S. ferus, S. gordonii, S. intermedius, S.
parasanguis, S. parauberis, S. salivarius, S. sanguis, S. suis) as
well as S. agalactiae were tested in the LightCycler with 0.07 ng
of genomic DNA per reaction. Only S. agalactiae yielded an
amplification signal, hence demonstrating that the assay is
species-specific. With the LightCycler.TM. assay using the internal
FRET probes, the detection limit for S. agalactiae was 1-2 genome
copies of genomic DNA.
Example 6
Specific Detection and Identification of Streptococcus agalactiae
Using atpD Sequences
[0272] As shown in Annex X, the comparison of atpD sequences from a
variety of bacterial species allowed the selection of PCR primers
specific for S. agalactiae. The primer design strategy is similar
to the strategy described in the preceding Example except that atpD
sequences were used in the alignment.
[0273] Four primers were selected, ASag42 (SEQ ID NO. 627), ASag52
(SEQ ID NO. 628), ASag206 (SEQ ID NO. 625) and ASag371 (SEQ ID NO.
626). The following combinations of these four primers give four
amplicons; SEQ ID NO. 627+SEQ ID NO. 625=190 bp, SEQ ID NO. 628+SEQ
ID NO. 625=180 bp, SEQ ID NO. 627+SEQ ID NO. 626=355 bp, and SEQ ID
NO. 628+SEQ ID NO. 626=345 bp.
[0274] Standard PCR was carried out on PTC-200 thermocyclers (MJ
Research Inc) using 0.4 .mu.M of each primers pair, 2.5 mM
MgCl.sub.2, BSA 0.05 mM, 1.times. taq Buffer (Promega), dNTP 0.2 mM
(Pharmacia), 0.5 U Taq DNA polymerase (Promega) coupled with
TaqStart.TM. antibody (Clontech Laboratories Inc., Palo Alto), 1
.mu.l of genomic DNA sample in a final volume of 20 .mu.L. The
optimal cycling conditions for maximum sensitivity and specificity
were adjusted for each primer pair. Three minutes at 95.degree. C.
for initial denaturation, then forty cycles of two steps consisting
of 1 second at 95.degree. C. and 30 seconds at the optimal
annealing temperature specified below were followed by terminal
extension at 72.degree. C. for 2 minutes. Detection of the PCR
products was made by electrophoresis in agarose gels (2%)
containing 0.25 .mu.g/ml of ethidium bromide. Since atpD sequences
are relatively more specific than tuf sequences, only the most
closely related species namely, the streptococcal species listed in
Table 9, were tested.
[0275] All four primer pairs only amplified the six S. agalactiae
strains. With an annealing temperature of 63.degree. C., the primer
pair SEQ ID NO. 627+SEQ ID NO. 625 had a sensitivity of 1-5 fg
(equivalent to 1-2 genome copies). At 55.degree. C., the primer
pair SEQ ID NO. 628+SEQ ID NO. 625 had a sensitivity of 2.5 fg
(equivalent to 1 genome copy). At 60.degree. C., the primer pair
SEQ ID NO. 627+SEQ ID NO. 626 had a sensitivity of 10 fg
(equivalent to 4 genome copies). At 58.degree. C., the primer pair
SEQ ID NO. 628+SEQ ID NO. 626 had a sensitivity of 2.5-5 fg
(equivalent to 1-2 genome copies). This proves that all four primer
pairs can detect S. agalactiae with high specificity and
sensitivity. Together with Example 5, this example demonstrates
that both tuf and atpD sequences are suitable and flexible targets
for the identification of microorganisms at the species level. The
fact that 4 different primer pairs based on atpD sequences led to
efficient and specific amplification of S. agalactiae demonstrates
that the challenge is to find target genes suitable for diagnostic
purposes, rather than finding primer pairs from these target
sequences.
Example 7
Development of a PCR Assay for Detection and Identification of
Staphylococci at Genus and Species Levels
Materials and Methods
[0276] Bacterial strains. The specificity of the PCR assay was
verified by using a panel of ATCC (America Type Culture Collection)
and DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen
GmbH; German Collection of Microorganisms and Cell Cultures)
reference strains consisting of 33 gram-negative and 47
gram-positive bacterial species (Table 12). In addition, 295
clinical isolates representing 11 different species of
staphylococci from the microbiology laboratory of the Centre
Hospitalier Universitaire de Quebec, Pavillon Centre Hospitalier de
l'Universite Laval (CHUL) (Step-Foy, Quebec, Canada) were also
tested to further validate the Staphylococcus-specific PCR assay.
These strains were all identified by using (i) conventional methods
or (ii) the automated MicroScan Autoscan-4 system equipped with the
Positive BP Combo Panel Type 6 (Dade Diagnostics, Mississauga,
Ontario, Canada). Bacterial strains from frozen stocks kept at
-80.degree. C. in brain heart infusion (BHI) broth containing 10%
glycerol were cultured on sheep blood agar or in BHI broth (Quelab
Laboratories Inc, Montreal, Quebec, Canada).
[0277] PCR primers and internal probes. Based on multiple sequence
alignments, regions of the tuf gene unique to staphylococci were
identified. Staphylococcus-specific PCR primers TStaG422 (SEQ ID
NO. 553) and TStaG765 (SEQ ID NO. 575) were derived from these
regions (Annex XII). These PCR primers are displaced by two
nucleotide positions compared to original Staphylococcus-specific
PCR primers described in our patent publication WO98/20157 (SEQ ID
NOs. 17 and 20 in the said patent publication). These modifications
were done to ensure specificity and ubiquity of the primer pair, in
the light of new tuf sequence data revealed in the present patent
application for several additional staphylococcal species and
strains.
[0278] Similarly, sequence alignment analysis were performed to
design genus and species-specific internal probes (see Annexes XIII
to XVI). Two internal probes specific for Staphylococcus (SEQ ID
NOs. 605-606), five specific for S. aureus (SEQ ID NOs. 584-588),
five specific for S. epidermidis (SEQ ID NO. 589-593), two specific
for S. haemolyticus (SEQ ID NOs. 594-595), three specific for S.
hominis (SEQ ID NOs. 596-598), four specific for S. saprophyticus
(SEQ ID NOs. 599-601 and 695), and two specific for
coagulase-negative Staphylococcus species including S. epidermidis,
S. hominis, S. saprophyticus, S. auricularis, S. capitis, S.
haemolyticus, S. lugdunensis, S. simulans, S. cohnii and S. warneri
(SEQ ID NOs. 1175-1176) were designed. The range of mismatches
between the Staphylococcus-specific 371-bp amplicon and each of the
20-mer species-specific internal probes was from 1 to 5, in the
middle of the probe when possible. No mismatches were present in
the two Staphylococcus-specific probes for the 11 species analyzed:
S. aureus, S. auricularis, S. capitis, S. cohnii, S. epidermidis,
S. haemolyticus, S. hominis, S. lugdunensis, S. saprophyticus, S.
simulans and S. warneri. In order to verify the intra-specific
sequence conservation of the nucleotide sequence, sequences were
obtained for the 371-bp amplicon from five unrelated ATCC and
clinical strains for each of the species S. aureus, S. epidermidis,
S. haemolyticus, S. hominis and S. saprophyticus. The Oligo.TM.
(version 5.0) primer analysis software (National Biosciences,
Plymouth, Minn.) was used to confirm the absence of
self-complementary regions within and between the primers or
probes. When required, the primers contained inosines or
degenerated nucleotides at one or more variable positions.
Oligonucleotide primers and probes were synthesized on a model 394
DNA synthesizer (Applied Biosystems, Mississauga, Ontario, Canada).
Detection of the hybridization was performed with the DIG-labeled
dUTP incorporated during amplification with the
Staphylococcus-specific PCR assay, and the hybridization signal was
detected with a luminometer (Dynex Technologies) as described above
in the section on luminescent detection of amplification products.
Annexes XIII to XVI illustrate the strategy for the selection of
several internal probes.
[0279] PCR amplification. For all bacterial species, amplification
was performed from purified genomic DNA or from a bacterial
suspension whose turbidity was adjusted to that of a 0.5 McFarland
standard, which corresponds to approximately 1.5.times.10.sup.8
bacteria per ml. One nanogram of genomic DNA or 1 .quadrature.l of
the standardized bacterial suspension was transferred directly to a
19 .quadrature.l PCR mixture. Each PCR reaction contained 50 mM
KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl.sub.2,
0.2 .quadrature.M (each) of the two Staphylococcus genus-specific
primers (SEQ ID NOs. 553 and 575), 200 .quadrature.M (each) of the
four deoxynucleoside triphosphates (Pharmacia Biotech), 3.3
.quadrature.g/.quadrature.l bovine serum albumin (BSA)
(Sigma-Aldrich Canada Ltd, Oakville, Ontario, Canada), and 0.5 U
Taq polymerase (Promega) coupled with TaqStart.TM. Antibody
(Clontech). The PCR amplification was performed as follows: 3 min.
at 94.degree. C. for initial denaturation, then forty cycles of two
steps consisting of 1 second at 95.degree. C. and 30 seconds at
55.degree. C., plus a terminal extension at 72.degree. C. for 2
minutes. Detection of the PCR products was made by electrophoresis
in agarose gels (2%) containing 0.25 .mu.g/ml of ethidium bromide.
Visualization of the PCR products was made under UV at 254 nm.
[0280] For determination of the sensitivities of the PCR assays,
two-fold dilutions of purified genomic DNA were used to determine
the minimal number of genome copies which can be detected.
Results
[0281] Amplifications with the Staphylococcus genus-specific PCR
assay. The specificity of the assay was assessed by performing
30-cycle and 40-cycle PCR amplifications with the panel of
gram-positive (47 species from 8 genera) and gram-negative (33
species from 22 genera) bacterial species listed in Table 12. The
PCR assay was able to detect efficiently 27 of 27 staphylococcal
species tested in both 30-cycle and 40-cycle regimens. For 30-cycle
PCR, all bacterial species tested other than staphylococci were
negative. For 40-cycle PCR, Enterococcus faecalis and Macrococcus
caseolyticus were slightly positive for the Staphylococcus-specific
PCR assay. The other species tested remained negative. Ubiquity
tests performed on a collection of 295 clinical isolates provided
by the microbiology laboratory of the Centre Hospitalier
Universitaire de Quebec, Pavillon Centre Hospitalier de
l'Universite Laval (CHUL), including Staphylococcus aureus (n=34),
S. auricularis (n=2), S. capitis (n=19), S. cohnii (n=5), S.
epidermidis (n=18), S. haemolyticus (n=21), S. hominis (n=73), S.
lugdunensis (n=17), S. saprophyticus (n=6), S. simulans (n=3), S.
warneri (n=32) and Staphylococcus sp. (n=65), showed a uniform
amplification signal with the 30-cycle PCR assays and a perfect
relation between the genotype and classical identification
schemes.
[0282] The sensitivity of the Staphylococcus-specific assay with
30-cycle and 40-cycle PCR protocols was determined by using
purified genomic DNA from the 11 staphylococcal species previously
mentioned. For PCR with 30 cycles, a detection limit of 50 copies
of genomic DNA was consistently obtained. In order to enhance the
sensitivity of the assay, the number of cycles was increased. For
40-cycle PCR assays, the detection limit was lowered to a range of
5-10 genome copies, depending on the staphylococcal species
tested.
[0283] Hybridization between the Staphylococcus-specific 371-bp
amplicon and species-specific or genus-specific internal probes.
Inter-species polymorphism was sufficient to generate
species-specific internal probes for each of the principal species
involved in human diseases (S. aureus, S. epidermidis, S.
haemolyticus, S. hominis and S. saprophyticus). In order to verify
the intra-species sequence conservation of the nucleotide sequence,
sequence comparisons were performed on the 371-bp amplicon from
five unrelated ATCC and clinical strains for each of the 5
principal staphylococcal species: S. aureus, S. epidermidis, S.
haemolyticus, S. hominis and S. saprophyticus. Results showed a
high level of conservation of nucleotide sequence between different
unrelated strains from the same species. This sequence information
allowed the development of staphylococcal species identification
assays using species-specific internal probes hybridizing to the
371-bp amplicon. These assays are specific and ubiquitous for those
five staphylococcal species. In addition to the species-specific
internal probes, the genus-specific internals probes were able to
recognize all or most Staphylococcus species tested.
Example 8
Differentiating Between the Two Closely Related Yeast Species
Candida albicans and Candida dubliniensis
[0284] It is often useful for the clinician to be able to
differentiate between two very closely related species of
microorganisms. Candida albicans is the most important cause of
invasive human mycose. In recent years, a very closely related
species, Candida dubliniensis, was isolated in immunosuppressed
patients. These two species are difficult to distinguish by classic
biochemical methods. This example demonstrates the use of tuf
sequences to differentiate Candida albicans and Candida
dubliniensis. PCR primers SEQ ID NOs. 11-12, from previous patent
publication WO98/20157, were selected for their ability to
specifically amplify a tuf (elongation factor 1 alpha type)
fragment from both species (see Annex XI for primer positions).
Within this tuf fragment, a region differentiating C. albicans and
C. dubliniensis by two nucleotides was selected and used to design
two internal probes (see Annex XI for probe design, SEQ ID NOs. 577
and 578) specific for each species. Amplification of genomic DNA
from C. albicans and C. dubliniensis was carried out using
DIG-11-dUTP as described above in the section on chemiluminescent
detection of amplification products. Internal probes SEQ ID NOs.
577 and 578 were immobilized on the bottom of individual microtiter
plates and hybridization was carried out as described above in the
above section on chemiluminescent detection of amplification
products. Luminometer data showed that the amplicon from C.
albicans hybridized only to probe SEQ ID NO. 577 while the amplicon
from C. dubliniensis hybridized only to probe SEQ ID NO. 578,
thereby demonstrating that each probe was species-specific.
Example 9
Specific Identification of Entamoeba histolytica
[0285] Upon analysis of tuf (elongation factor 1 alpha) sequence
data, it was possible to find four regions where Entamoeba
histolytica sequences remained conserved while other parasitical
and eukaryotic species have diverged. Primers TEntG38 (SEQ ID NO.
703), TEntG442 (SEQ ID NO. 704), TEntG534 (SEQ ID NO. 705), and
TEntG768 (SEQ ID NO. 706) were designed so that SEQ ID NO. 703
could be paired with the three other primers. On PTC-200
thermocyclers (MJ Research), the cycling conditions for initial
sensitivity and specificity testing were 3 min. at 94.degree. C.
for initial denaturation, then forty cycles of two steps consisting
of 1 second at 95.degree. C. and 30 seconds at 55.degree. C.,
followed by terminal extension at 72.degree. C. for 2 minutes.
Detection of the PCR products was made by electrophoresis in
agarose gels (2%) containing 0.25 .mu.g/ml of ethidium bromide. The
three primer pairs could detect the equivalent of less than 200 E.
histolytica genome copies. Specificity was tested using 0.5 ng of
purified genomic DNA from a panel of microorganisms including
Babesia bovis, Babesia microtti, Candida albicans, Crithidia
fasciculata, Leishmania major, Leishmania hertigi and Neospora
caninum. Only E. histolytica DNA could be amplified, thereby
suggesting that the assay was species-specific.
Example 10
Sensitive Identification of Chlamydia trachomatis
[0286] Upon analysis of tuf sequence data, it was possible to find
two regions where Chlamydia trachomatis sequences remained
conserved while other species have diverged. Primers Ctr82 (SEQ ID
NO. 554) and Ctr249 (SEQ ID NO. 555) were designed. With the
PTC-200 thermocyclers (MJ Research), the optimal cycling conditions
for maximum sensitivity and specificity were determined to be 3
min. at 94.degree. C. for initial denaturation, then forty cycles
of two steps consisting of 1 second at 95.degree. C. and 30 seconds
at 60.degree. C., followed by terminal extension at 72.degree. C.
for 2 minutes. Detection of the PCR products was made by
electrophoresis in agarose gels (2%) containing 0.25 .mu.g/ml of
ethidium bromide. The assay could detect the equivalent of 8 C.
trachomatis genome copies. Specificity was tested with 0.1 ng of
purified genomic DNA from a panel of microorganisms including 22
species commonly encountered in the vaginal flora (Bacillus
subtilis, Bacteroides fragilis, Candida albicans, Clostridium
difficile, Corynebacterium cervicis, Corynebacterium urealyticum,
Enterococcus faecalis, Enterococcus faecium, Escherichia coli,
Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus
influenzae, Klebsiella oxytoca, Lactobacillus acidophilus,
Peptococcus niger, Peptostreptococcus prevotii, Porphyromonas
asaccharolytica, Prevotella melaminogenica, Propionibacterium
acnes, Staphylococcus aureus, Streptococcus acidominimus, and
Streptococcus agalactiae). Only C. trachomatis DNA could be
amplified, thereby suggesting that the assay was
species-specific.
Example 11
Genus-Specific Detection and Identification of Enterococci
[0287] Upon analysis of tuf sequence data and comparison with the
repertory of tuf sequences, it was possible to find two regions
where Enterococcus sequences remained conserved while other genera
have diverged (Annex XVII). Primer pair Encg313dF and Encg599c (SEQ
ID NOs. 1137 and 1136) was tested for its specificity by using
purified genomic DNA from a panel of bacteria listed in Table 10.
Using the PTC-200 thermocycler (MJ Research), the optimal cycling
conditions for maximum sensitivity and specificity were determined
to be 3 min. at 94.degree. C. for initial denaturation, then forty
cycles of two steps consisting of 1 second at 95.degree. C. and 30
seconds at 55.degree. C., followed by terminal extension at
72.degree. C. for 2 minutes. Detection of the PCR products was made
by electrophoresis in agarose gels (2%) containing 0.25 .mu.g/ml of
ethidium bromide. Visualization of the PCR products was made under
UV at 254 nm. The 18 enterococcal species listed in Table 10 were
all amplified efficiently. The only other species amplified were
Abiotrophia adiacens, Gemella haemolysans and Gemella morbillorum,
three gram-positive species. Sensitivity tested with several
strains of E. casseliflavus, E. faecium, E. faecalis, E. flavescens
and E. gallinarum and with one strain of each other Enterococcus
species listed in Table 10 ranged from 1 to 10 copies of genomic
DNA. The sequence variation within the 308-bp amplicon was
sufficient so that internal probes could be used to speciate the
amplicon and differentiate enterococci from Abiotrophia adiacens,
Gemella haemolysans and Gemella morbillorum, thereby allowing to
achieve excellent specificity. Species-specific internal probes
were generated for each of the clinically important species, E.
faecalis (SEQ ID NO. 1174), E. faecium (SEQ ID NO. 602), and the
group including E. casseliflavus, E. flavescens and E. gallinarum
(SEQ ID NO. 1122) (Annex XVIII). The species-specific internal
probes were able to differentiate their respective Enterococcus
species from all other Enterococcus species. These assays are
sensitive, specific and ubiquitous for those five Enterococcus
species.
Example 12
Identification of the Major Bacterial Platelets Contaminants Using
Tuf Sequences with a Multiplex PCR Test
[0288] Blood platelets preparations need to be monitored for
bacterial contaminations. The tuf sequences of 17 important
bacterial contaminants of platelets were aligned. As shown in Annex
XIX, analysis of these sequences allowed the design of PCR primers.
Since in the case of contamination of platelet concentrates,
detecting all species (not just the more frequently encountered
ones) is desirable, perfect specificity of primers was not an issue
in the design. However, sensitivity is important. That is why, to
avoid having to put too much degeneracy, only the most frequent
contaminants were included in primer design, knowing that the
selected primers would anyway be able to amplify more species than
the 17 used in the design because they target highly conserved
regions of tuf sequences. Oligonucleotide sequences which are
conserved in these 17 major bacterial contaminants of platelet
concentrates were chosen (oligos Tplaq 769 and Tplaq 991,
respectively SEQ ID NOs. 636 and 637) thereby permitting the
detection of these bacterial species. However, sensitivity was
slightly deficient with staphylococci. To ensure maximal
sensitivity in the detection of all the more frequent bacterial
contaminants, a multiplex assay also including oligonucleotide
primers targeting the Staphylococcus genera (oligos Stag 422, SEQ
ID NO. 553; and Stag 765, SEQ ID NO. 575) was developed. The
bacterial species detected with the assay are listed in Table
14.
[0289] The primer pairs, oligos SEQ ID NO. 636 and SEQ ID NO. 637
that give an amplification product of 245 pb, and oligos SEQ ID NO.
553 and SEQ ID NO. 575 that give an amplification product of 368
pb, were used simultaneously in the multiplex PCR assay. Detection
of these PCR products was made on the LightCycler thermocycler
(Idaho Technology) using SYBR.RTM. Green I (Molecular Probe Inc.).
SYBR.RTM. Green I is a fluorescent dye that binds specifically to
double-stranded DNA.
[0290] Fluorogenic detection of PCR products with the LightCycler
was carried out using 1.0 .mu.M of both Tplaq primers (SEQ ID NOs.
636-637) and 0.4 .mu.M of both TStaG primers (SEQ ID NOs. 553 and
575), 2.5 mM MgCl.sub.2, BSA 7.5 .mu.M, dNTP 0.2 mM (Pharmacia), 10
mM Tris-HCl (pH 8.3), 50 mM KCl, 0.5 U Taq DNA polymerase
(Boerhinger Mannheim) coupled with TaqStart.TM. antibody
(Clontech), and 0.07 ng of genomic DNA sample in a final volume of
7 .mu.l. The optimal cycling conditions for maximum sensitivity and
specificity were 1 minute at 94.degree. C. for initial
denaturation, then forty-five cycles of three steps consisting of 0
second at 95.degree. C., 5 seconds at 60.degree. C. and 9 seconds
at 72.degree. C. Amplification was monitored during each elongation
cycle by measuring the level of SYBR.RTM. Green I. However, real
analysis takes place after PCR. Melting curves are done for each
sample and transformation of the melting peak allows determination
of Tm. Thus primer-dimer and specific PCR product are
discriminated. With this assay, all prominent bacterial
contaminants of platelet concentrates listed in Annex XIX and Table
14 were detected. Sensitivity tests were performed on the 9 most
frequent bacterial contaminants of platelets. The detection limit
was less than 20 genome copies for E. cloacae, B. cereus, S.
choleraesuis and S. marcescens; less than 15 genome copies for P.
aeruginosa; and 2 to 3 copies were detected for S. aureus, S.
epidermidis, E. coli and K. pneumoniae. Further refinements of
assay conditions should increase sensitivity levels.
Example 13
The Resolving Power of the tuf and atpD Sequences Databases is
Comparable to the Biochemical Methods for Bacterial
Identification
[0291] The present gold standard for bacterial identification is
mainly based on key morphological traits and batteries of
biochemical tests. Here we demonstrate that the use of tuf and atpD
sequences combined with simple phylogenetic analysis of databases
formed by these sequences is comparable to the gold standard. In
the process of acquiring data for the tuf sequences, we sequenced
the tuf gene of a strain that was given to us labelled as
Staphylococcus hominis ATCC 35982. That tuf sequence (SEQ ID NO.
192) was incorporated into the tuf sequences database and subjected
to a basic phylogenic analysis using the Pileup command from
version 10 of the GCG package (Genetics Computer Group). This
analysis indicated that SEQ ID NO. 192 is not associated with other
S. hominis strains but rather with the S. warneri strains. The ATCC
35982 strain was sent to the reference laboratory of the
Laboratoire de sante publique du Quebec (LSPQ). They used the
classic identification scheme for staphylococci (Kloos and
Schleifer, 1975., J. Clin. Microbiol. 1:82-88). Their results shown
that although the colonial morphology could correspond to S.
hominis, the more precise biochemical assays did not. These assays
included discriminant mannitol, mannose and ribose acidification
tests as well as rapid and dense growth in deep thioglycolate agar.
The LSPQ report identified strain ATCC 35982 as S. warneri which
confirms our database analysis. The same thing happened for S.
warneri (SEQ ID NO. 187) which had initially been identified as S.
haemolyticus by a routine clinical laboratory using a low resolving
power automated system (MicroScan, AutoScan-4.TM.). Again, the tuf
and LSPQ analysis agreed on its identification as S. warneri. In
numerous other instances, in the course of acquiring tuf and atpD
sequence data from various species and genera, analysis of our tuf
and/or atpD sequence databases permitted the exact identification
of mislabeled or erroneously identified strains. These results
clearly demonstrate the usefulness and the high resolving power of
our sequence-based identification assays using the tuf and atpD
sequences databases.
Example 14
Detection of Group B Streptococci from Clinical Specimens
Introduction
[0292] Streptococcus agalactiae, the group B streptococcus (GBS),
is responsible for a severe illness affecting neonate infants. The
bacterium is passed from the healthy carrier mother to the baby
during delivery. To prevent this infection, it is recommended to
treat expectant mothers susceptible of carrying GBS in their
vaginal/anal flora. Carrier status is often a transient condition
and rigorous monitoring requires cultures and classic bacterial
identification weeks before delivery. To improve the detection and
identification of GBS we developed a rapid, specific and sensitive
PCR test fast enough to be performed right at delivery.
Materials and Methods
[0293] GBS clinical specimens. A total of 66 duplicate vaginal/anal
swabs were collected from 41 consenting pregnant women admitted for
delivery at the Centre Hospitalier Universitaire de Quebec,
Pavillon Saint-Francois d'Assise following the CDC recommendations.
The samples were obtained either before or after rupture of
membranes. The swab samples were tested at the Centre de Recherche
en Infectiologie de l'Universite Laval within 24 hours of
collection. Upon receipt, one swab was cut and then the tip of the
swab was added to GNS selective broth for identification of group B
streptococci (GBS) by the standard culture methods recommended by
the CDC. The other swab was processed following the instruction of
the IDI DNA extraction kit (Infectio Diagnotics (IDI) Inc.) prior
to PCR amplification.
[0294] Oligonucleotides. PCR primers, Tsag340 (SEQ ID NO. 549) and
Tsag552 (SEQ ID NO. 550) complementary to the regions of the tuf
gene unique for GBS were designed based upon a multiple sequence
alignment using our repertory of tuf sequences. Oligo primer
analysis software (version 5.0) (National Biosciences) was used to
analyse primers annealing temperature, secondary structure
potential as well as mispriming and dimerization potential. The
primers were synthesized using a model 391 DNA synthesizer (Applied
Biosystems).
[0295] A pair of fluorescently labeled adjacent hybridization
probes Sag465-F (SEQ ID NO. 583) and Sag436-C (SEQ ID NO. 582) were
synthesized and purified by Operon Technologies. They were designed
to meet the recommendations of the manufacturer (Idaho Technology)
and based upon multiple sequence alignment analysis using our
repertory of tuf sequences to be specific and ubiquitous for GBS.
These adjacent probes, which are separated by one nucleotide, allow
fluorescence resonance energy transfer (FRET), generating an
increased fluorescence signal when both hybridized simultaneously
to their target sequences. The probe SEQ ID NO. 583 was labeled
with FITC in 3 prime while SEQ ID NO. 582 was labeled with Cy5 in 5
prime. The Cy5-labeled probes contained a 3'-blocking phosphate
group to prevent extension of the probes during the PCR
reactions.
[0296] PCR amplification. Conventional amplifications were
performed either from 2 .mu.l of a purified genomic DNA preparation
or cell lysates of vaginal/anal specimens. The 20 .mu.l PCR mixture
contained 0.4 .mu.M of each GBS-specific primer (SEQ ID NOs.
549-550), 200 .mu.M of each deoxyribonucleotide (Pharmacia
Biotech), 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100,
2.5 mM MgCl.sub.2, 3.3 mg/ml bovine serum albumin (BSA) (Sigma),
and 0.5 U of Taq polymerase (Promega) combined with the
TaqStart.TM. antibody (Clontech). The TaqStart.TM. antibody, which
is a neutralizing monoclonal antibody of Taq DNA polymerase, was
added to all PCR reactions to enhance the efficiency of the
amplification. The PCR mixtures were subjected to thermal cycling
(3 min at 95.degree. C. and then 40 cycles of 1 s at 95.degree. C.,
and 30 s at 62.degree. C. with a 2-min final extension at
72.degree. C.) with a PTC-200 DNA Engine thermocycler (MJ
research). The PCR-amplified reaction mixture was resolved by
agarose gel electrophoresis.
[0297] The LightCycler.TM. PCR amplifications were performed with 1
.mu.l of a purified genomic DNA preparation or cell lysates of
vaginal/anal specimens. The 10 .mu.l amplification mixture
consisted of 0.4 .mu.M each GBS-specific primer (SEQ ID NOs.
549-550), 200 .mu.M each dNTP, 0.2 .mu.M each fluorescently labeled
probe (SEQ ID NOs. 582-583), 300 .mu.g/ml BSA (Sigma), and 1 .mu.l
of 10.times.PC2 buffer (containing 50 mM Tris-HCl (pH 9.1), 16 mM
ammonium sulfate, 3.5 mM Mg.sup.2+, and 150 .mu.g/ml BSA) and 0.5 U
KlenTaq1.TM. (AB Peptides) coupled with TaqStart.TM. antibody
(Clontech). KlenTaq1.TM. is a highly active and more heat-stable
DNA polymerase without 5'-exonuclease activity. This prevents
hydrolysis of hybridized probes by the 5' to 3' exonuclease
activity. A volume of 7 .mu.l of the PCR mixture was transferred
into a composite capillary tube (Idaho Technology). The tubes were
then centrifuged to move the reaction mixture to the tips of the
capillaries and then cleaned with optical-grade methanol.
Subsequently the capillaries were loaded into the carousel of a
LC32 LightCycler.TM. (Idaho Technology), an instrument that
combines rapid-cycle PCR with fluorescence analysis for continuous
monitoring during amplification. The PCR reaction mixtures were
subjected to a denaturation step at 94.degree. C. for 3 min
followed by 45 cycles of 0 s at 94.degree. C., 20 s at 64.degree.
C. and 10 s at 72.degree. C. with a temperature transition rate of
20.degree. C./s. Fluorescence signals were obtained at each cycle
by sequentially positioning each capillary on the carousel at the
focus of optical elements affiliated to the built-in fluorimeter
for 100 milliseconds. Complete amplification and analysis required
about 35 min.
[0298] Specificity and sensitivity tests. The specificity of the
conventional and LightCycler.TM. PCR assays was verified by using
purified genomic DNA (0.1 ng/reaction) from a battery of ATCC
reference strains representing 35 clinically relevant gram-positive
species (Abiotrophia defectiva ATCC 49176, Bifidobacterium breve
ATCC 15700, Clostridium difficile ATCC 9689, Corynebacterium
urealyticum ATCC 43042, Enterococcus casseliflavus ATCC 25788,
Enterococcus durans ATCC 19432, Enterococcus faecalis ATCC 29212,
Enterococcus faecium ATCC 19434, Enterococcus gallinarum ATCC
49573, Enterococcus raffinosus ATCC 49427, Lactobacillus reuteri
ATCC 23273, Lactococcus lactis ATCC 19435, Listeria monocytogenes
ATCC 15313, Peptococcus niger ATCC 27731, Peptostreptococcus
anaerobius ATCC 27337, Peptostreptococcus prevotii ATCC 9321,
Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC
14990, Staphylococcus haemolyticus ATCC 29970, Staphylococcus
saprophyticus ATCC 15305, Streptococcus agalactiae ATCC 27591,
Streptococcus anginosus ATCC 33397, Streptococcus bovis ATCC 33317,
Streptococcus constellatus ATCC 27823, Streptococcus dysgalactiae
ATCC 43078, Streptococcus gordonii ATCC 10558, Streptococcus mitis
ATCC 33399, Streptococcus mutans ATCC 25175, Streptococcus oralis
ATCC 35037, Streptococcus parauberis ATCC 6631, Streptococcus
pneumoniae ATCC 6303, Streptococcus pyogenes ATCC 19615,
Streptococcus salivarius ATCC 7073, Streptococcus sanguinis ATCC
10556, Streptococcus uberis ATCC 19436). These microbial species
included 15 species of streptococci and many members of the normal
vaginal and anal floras. In addition, 40 GBS isolates of human
origin, whose identification was confirmed by a latex agglutination
test (Streptex, Murex), were also used to evaluate the ubiquity of
the assay.
[0299] For determination of the sensitivities (i.e., the minimal
number of genome copies that could be detected) for conventional
and LightCycler.TM. PCR assays, serial 10-fold or 2-fold dilutions
of purified genomic DNA from 5 GBS ATCC strains were used.
Results
[0300] Evaluation of the GBS-specific conventional and
LightCycler.TM. PCR assays. The specificity of the two assays
demonstrated that only DNAs from GBS strains could be amplified.
Both PCR assays did not amplify DNAs from any other bacterial
species tested including 14 streptococcal species other than GBS as
well as phylogenetically related species belonging to the genera
Enterococcus, Peptostreptococcus and Lactococcus. Important members
of the vaginal or anal flora, including coagulase-negative
staphylococci, Lactobacillus sp., and Bacteriodes sp. were also
negative with the GBS-specific PCR assay. The LightCycler.TM. PCR
assays detected only GBS DNA by producing an increased fluorescence
signal which was interpreted as a positive PCR result. Both PCR
methods were able to amplify all of 40 GBS clinical isolates,
showing a perfect correlation with the phenotypic identification
methods.
[0301] The sensitivity of the assay was determined by using
purified genomic DNA from the 5 ATCC strains of GBS. The detection
limit for all of these 5 strains was one genome copy of GBS. The
detection limit of the assay with the LightCycler.TM. was 3.5 fg of
genomic DNA (corresponding to 1-2 genome copies of GBS). These
results confirmed the high sensitivity of our GBS-specific PCR
assay.
[0302] Direct Detection of GBS from vaginal/anal specimens. Among
66 vaginal/anal specimens tested, 11 were positive for GBS by both
culture and PCR. There was one sample positive by culture only. The
sensitivity of both PCR methods with vaginal/anal specimens for
identifying colonization status in pregnant women at delivery was
91.7% when compared to culture results. The specificity and
positive predictive values were both 100% and the negative
predictive value was 97.8%. The time for obtaining results was
approximately 45 min for LightCycler.TM. PCR, approximately 100 min
for conventional PCR and 48 hours for culture.
Conclusion
[0303] We have developed two PCR assays (conventional and
LightCycler.TM.) for the detection of GBS, which are specific
(i.e., no amplification of DNA from a variety of bacterial species
other than GBS) and sensitive (i.e., able to detect around 1 genome
copy for several reference ATCC strains of GBS). Both PCR assays
are able to detect GBS directly from vaginal/anal specimens in a
very short turnaround time. Using the real-time PCR assay on
LightCycler.TM., we can detect GBS carriage in pregnant women at
delivery within 45 minutes.
Example 15
Simultaneous Detection and Identification of Streptococcus pyogenes
and its Pyrogenic Exotoxin A
[0304] The rapid detection of Streptococcus pyogenes and of its
pyrogenic exotoxin A is of clinical importance. We developed a
multiplex assay which permits the detection of strains of S.
pyogenes carrying the pyrogenic toxin A gene, which is associated
with scarlet fever and other pathologies. In order to specifically
detect S. pyogenes, nucleotide sequences of the pyrrolidone
carboxylyl peptidase (pcp) gene were aligned to design PCR primers
Spy291 (SEQ ID NO. 1211) and Spy473 (SEQ ID NO. 1210). Next, we
designed primers for the specific detection of the pyrogenic
exotoxin A. Nucleotide sequences of the speA gene, carried on the
bacteriophage T12, were aligned as shown in Annex XXIII to design
PCR primers Spytx814 (SEQ ID NO. 994) and Spytx 927 (SEQ ID NO.
995).
[0305] The primer pairs: oligos SEQ ID NOs. 1210-1211, yielding an
amplification product of 207 bp, and oligos SEQ ID NOs. 994-995,
yielding an amplification product of 135 bp, were used in a
multiplex PCR assay.
[0306] PCR amplification was carried out using 0.4 .mu.M of both
pairs of primers, 2.5 mM MgCl.sub.2, BSA 0.05 .mu.M, dNTP 0.2 .mu.M
(Pharmacia), 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM
MgCl.sub.2, 0.5 U Taq DNA polymerase (Promega) coupled with
TaqStart.TM. antibody (Clontech Laboratories Inc.), and 1 .mu.l of
genomic DNA sample in a final volume of 20 PCR amplification was
performed using a PTC-200 thermal cycler (MJ Research). The optimal
cycling conditions for maximum specificity and sensitivity were 3
minutes at 94.degree. C. for initial denaturation, then forty
cycles of two steps consisting of 1 second at 95.degree. C. and 30
seconds at 63.degree. C., followed by a final step of 2 minutes at
72.degree. C. Detection of the PCR products was made by
electrophoresis in agarose gels (2%) containing 0.25 .mu.g/ml of
ethidium bromide. Visualization of the PCR products was made under
UV at 254 nm.
[0307] The detection limit was less than 5 genome copies for both
S. pyogenes and its pyrogenic exotoxin A. The assay was specific
for pyrogenic exotoxin A-producing S. pyogenes: strains of the 27
other species of Streptococcus tested, as well as 20 strains of
various gram-positive and gram-negative bacterial species were all
negative.
[0308] A similar approach was used to design an alternative set of
speA-specific primers (SEQ ID NOs. 996 to 998, see Annex XXIV). In
addition, another set of primers based on the tuf gene (SEQ ID NOs.
999 to 1001, see Annex XXV) could be used to specifically detect
Streptococcus pyogenes.
Example 16
Real-Time Detection and Identification of Shiga Toxin-Producing
Bacteria
[0309] Shiga toxin-producing Escherichia coli and Shigella
dysenteriae cause bloody diarrhea. Currently, identification relies
mainly on the phenotypic identification of S. dysenteriae and E.
coli serotype O157:H7. However, other serotypes of E. coli are
increasingly found to be producers of type 1 and/or type 2 Shiga
toxins. Two pairs of PCR primers targeting highly conserved regions
present in each of the Shiga toxin genes stx.sub.1 and stx.sub.2
were designed to amplify all variants of those genes (see Annexes
XXVI and XXVII). The first primer pair, oligonucleotides 1SLT224
(SEQ ID NO. 1081) and 1SLT385 (SEQ ID NO. 1080), yields an
amplification product of 186 by from the six, gene. For this
amplicon, the 1SLTB1-Fam (SEQ ID NO. 1084) molecular beacon was
designed for the specific detection of six, using the fluorescent
label 6-carboxy-fluorescein. The 1 SltS1-FAM (SEQ ID NO. 2012)
molecular scorpion was also designed as an alternate way for the
specific detection of stx.sub.1. A second pair of PCR primers,
oligonucleotides 2SLT537 (SEQ ID NO. 1078) and 2SLT678b (SEQ ID NO.
1079), yields an amplification product of 160 by from the stx.sub.2
gene. Molecular beacon 2SLTB1-Tet (SEQ ID NO. 1085) was designed
for the specific detection of stx.sub.2 using the fluorescent label
5-tetrachloro-fluorescein. Both primer pairs were combined in a
multiplex PCR assay.
[0310] PCR amplification was carried out using 0.8 .mu.M of primer
pair SEQ ID NOs. 1080-1081, 0.5 .mu.M of primer pair SEQ ID NOs.
1078-1079, 0.3 .mu.M of each molecular beacon, 8 mM MgCl.sub.2, 490
.mu.g/mL BSA, 0.2 mM dNTPs (Pharmacia), 50 mM Tris-HCl, 16 mM
NH.sub.4SO.sub.4, 1.times. TaqMaster (Eppendorf), 2.5 U KlenTaq1
DNA polymerase (AB Peptides) coupled with TaqStart.TM. antibody
(Clontech Laboratories Inc.), and 1 .mu.l of genomic DNA sample in
a final volume of 25 PCR amplification was performed using a
SmartCycler thermal cycler (Cepheid). The optimal cycling
conditions for maximum sensitivity and specificity were 60 seconds
at 95.degree. C. for initial denaturation, then 45 cycles of three
steps consisting of 10 seconds at 95.degree. C., 15 seconds at
56.degree. C. and 5 seconds at 72.degree. C. Detection of the PCR
products was made in real-time by measuring the fluorescent signal
emitted by the molecular beacon when it hybridizes to its target at
the end of the annealing step at 56.degree. C.
[0311] The detection limit was the equivalent of less than 5 genome
copies. The assay was specific for the detection of both toxins, as
demonstrated by the perfect correlation between PCR results and the
phenotypic characterization performed using antibodies specific for
each Shiga toxin type. The assay was successfully performed on
several Shiga toxin-producing strains isolated from various
geographic areas of the world, including 10 O157:H7 E. coli, 5
non-O157:H7 E. coli and 4 S. dysenteriae.
Example 17
Development of a PCR Assay for the Detection and Identification of
Staphylococci at Genus and Species Levels and its Associated mecA
Gene
[0312] The Staphylococcus-specific PCR primers described in Example
7 (SEQ ID NOs. 553 and 575) were used in multiplex with the
mecA-specific PCR primers and the S. aureus-specific primers
described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 261
and 262 for mecA and SEQ ID NOs. 152 and 153 for S. aureus in the
said patent). Sequence alignment analysis of 10 publicly available
mecA gene sequences allowed to design an internal probe specific to
mecA (SEQ ID NO. 1177). An internal probe was also designed for the
S. aureus-specific amplicon (SEQ ID NO 1234). PCR amplification and
agarose gel electrophoresis of the amplified products were
performed as described in Example 7, with the exception that 0.4
.mu.M (each) of the two Staphylococcus-specific primers (SEQ ID
NOs. 553 and 575) and 0.4 .mu.M (each) of the mecA-specific primers
and 0.4 .mu.M (each) of the S. aureus-specific primers were used in
the PCR mixture. The specificity of the multiplex assay with
40-cycle PCR protocols was verified by using purified genomic DNA
from five methicillin-resistant and fifteen methicillin-sensitive
staphylococcal strains. The sensitivity of the multiplex assay with
40-cycle PCR protocols was determined by using purified genomic DNA
from twenty-three methicillin-resistant and twenty-eight
methicillin-sensitive staphylococcal strains. The detection limit
was 2 to 10 genome copies of genomic DNA, depending on the
staphylococcal species tested. Furthermore, the mecA-specific
internal probe, the S. aureus-specific internal probe and the
coagulase-negative staphylococci-specific internal probe (described
in Example 7) were able to recognize twenty-three
methicillin-resistant staphylococcal strains and twenty-eight
methicillin-sensitive staphylococcal strains with high sensitivity
and specificity.
[0313] The format of the assay is not limited to the one described
above. A person skilled in the art could adapt the assay for
different formats such as PCR with real-time detection using
molecular beacon probes. Molecular beacon probes designed to be
used in this assay include, but are not limited to, SEQ ID NO. 1232
for detection of the S. aureus-specific amplicon, SEQ ID NO. 1233
for detection of coagulase-negative staphylococci and SEQ ID NO.
1231 for detection of mecA.
[0314] Alternatively, a multiplex PCR assay containing the
Staphylococcus-specific PCR primers described in Example 7 (SEQ ID
NOs. 553 and 575) and the mecA-specific PCR primers described in
our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 261 and 262 in
the said patent) were developed. PCR amplification and agarose gel
electrophoresis of the amplified products were performed as
described in Example 7, with the exception that 0.4 .mu.M (each) of
the Staphylococcus-specific primers (SEQ ID NOs. 553 and 575) and
0.4 .mu.M (each) of the mecA-specific primers described in our
assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 261 and 262 in the
said patent) were used in the PCR mixture. The sensitivity of the
multiplex assay with 40-cycle PCR protocols was determined by using
purified genomic DNA from two methicillin-resistant and five
methicillin-sensitive staphylococcal strains. The detection limit
was 2 to 5 copies of genomic DNA, depending on the staphylococcal
species tested. The specificity of the multiplex PCR assay coupled
with capture-probe hybridization was tested with two strains of
methicillin-resistant S. aureus, two strains of
methicillin-sensitive S. aureus and seven strains of
methicillin-sensitive coagulase-negative staphylococci. The
mecA-specific internal probe (SEQ ID NO. 1177) and the S.
aureus-specific internal probe (SEQ ID NO. 587) described in
Example 7 were able to recognize all the strains with high
specificity showing a perfect correlation with susceptibility to
methicillin. The sensitivity of the PCR assay coupled with
capture-probe hybridization was tested with one strain of
methicillin-resistant S. aureus. The detection limit was around 10
copies of genomic DNA.
Example 18
Sequencing of pbp1a, pbp2b and pbp2x Genes of Streptococcus
pneumoniae
[0315] Penicillin resistance in Streptococcus pneumoniae involves
the sequential alteration of up to five penicillin-binding proteins
(PBPs) 1A, 1B, 2A, 2X and 2B in such a way that their affinity is
greatly reduce toward the antibiotic molecule. The altered PBP
genes have arisen as the result of interspecies recombination
events from related streptococcal species. Among the PBPs usually
found in S. pneumoniae, PBPs 1A, 2B, and 2X play the most important
role in the development of penicillin resistance. Alterations in
PBP 2B and 2X mediate low-level resistance to penicillin while
additional alterations in PBP 1A plays a significant role in full
penicillin resistance.
[0316] In order to generate a database for pbp sequences that can
be used for design of primers and/or probes for the specific and
ubiquitous detection of .beta.-lactam resistance in S. pneumoniae,
pbp1a, pbp2b and pbp2x DNA fragments sequenced by us or selected
from public databases (GenBank and EMBL) from a variety of S.
pneumoniae strains were used to design oligonucleotide primers.
This database is essential for the design of specific and
ubiquitous primers and/or probes for detection of .beta.-lactam
resistance in S. pneumoniae since the altered PBP 1A, PBP 2B and
PBP 2X of .beta.-lactam resistant S. pneumoniae are encoded by
mosaic genes with numerous sequence variations among resistant
isolates. The PCR primers were located in conserved regions of pbp
genes and were able to amplify pbp1a, pbp2b, and pbp2x sequences of
several strains of S. pneumoniae having various levels of
resistance to penicillin and third-generation cephalosporins. Using
primer pairs SEQ ID NOs. 1125 and 1126, SEQ ID NOs. 1142 and 1143,
SEQ ID NOs. 1146 and 1147, it was possible to amplify and determine
pbp1a sequences SEQ ID NOs. 1004-1018, 1648, 2056-2060 and
2062-2064, pbp2b sequences SEQ ID NOs. 1019-1033, and pbp2x
sequences SEQ ID NOs. 1034-1048. Six other PCR primers (SEQ ID NOs.
1127-1128, 1144-1145, 1148-1149) were also designed and used to
complete the sequencing of pbp1a, pbp2b and pbp2x amplification
products. The described primers (SEQ ID NOs. 1125 and 1126, SEQ ID
NOs. 1142 and 1143, SEQ ID NOs. 1146 and 1147, SEQ ID NOs.
1127-1128, 1144-1145, 1148-1149) represent a powerful tool for
generating new pbp sequences for design of primers and/or probes
for detection of .beta.-lactam resistance in S. pneumoniae.
Example 19
Sequencing of hexA Genes of Streptococcus Species
[0317] The hexA sequence of S. pneumoniae described in our assigned
U.S. Pat. No. 5,994,066 (SEQ ID NO. 31 in the said patent, SEQ ID
NO. 1183 in the present application) allowed the design of a PCR
primer (SEQ ID NO. 1182) which was used with primer Spn1401
described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NO. 156
in the said patent, SEQ ID NO. 1179 in the present application) to
generate a database for hexA sequences that can be used to design
primers and/or probes for the specific identification and detection
of S. pneumoniae (Annex XLII). Using primers SEQ ID NO. 1179 and
SEQ ID NO. 1182 (Annex XLII), it was possible to amplify and
determine the hexA sequence from S. pneumoniae (4 strains) (SEQ ID
NOs. 1184-1187), S. mitis (three strains) (SEQ ID NOs. 1189-1191)
and S. oralis (SEQ ID NO. 1188).
Example 20
Development of Multiplex PCR Assays Coupled with Capture Probe
Hybridization for the Detection and Identification of Streptococcus
pneumoniae and its Penicillin Resistance Genes
[0318] Two different assays were developed to identify S.
pneumoniae and its susceptibility to penicillin.
Assay I:
[0319] Bacterial strains. The specificity of the multiplex PCR
assay was verified by using a panel of ATCC (American Type Culture
Collection) reference strains consisting of 33 gram-negative and 67
gram-positive bacterial species (Table 13). In addition, a total of
98 strains of S. pneumoniae, 16 strains of S. mitis and 3 strains
of S. oralis from the American Type Culture Collection, the
microbiology laboratory of the Centre Hospitalier Universitaire de
Quebec, Pavillon Centre Hospitalier de l'Universite Laval (CHUL),
(Step-Foy, Quebec, Canada), the Laboratoire de sante publique du
Quebec, (Sainte-Anne-de-Bellevue, Quebec, Canada), the Sunnybrook
and Women's College Health Sciences Centre (Toronto, Canada), the
Infectious Diseases Section, Department of Veterans Affairs Medical
Center, (Houston, USA) were also tested to further validate the
Streptococcus pneumoniae-specific PCR assay. The penicillin MICs
(minimal inhibitory concentrations) were measured by the broth
dilution method according to the recommended protocol of NCCLS.
[0320] PCR primers and internal probes. The analysis of hexA
sequences from a variety of streptococcal species from the publicly
available hexA sequence and from the database described in Example
19 (SEQ ID NOs. 1184-1191) allowed the selection of a PCR primer
specific to S. pneumoniae, SEQ ID NO. 1181. This primer was used
with the S. pneumoniae-specific primer SEQ ID NO. 1179 to generate
an amplification product of 241 by (Annex XLII). The PCR primer SEQ
ID NO. 1181 is located 127 nucleotides downstream on the hexA
sequence compared to the original S. pneumoniae-specific PCR primer
Spn1515 described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID
NO. 157 in the said patent). These modifications were done to
ensure the design of the S. pneumoniae-specific internal probe
according to the new hexA sequences of several streptococcal
species from the database described in Example 19 (SEQ ID NOs.
1184-1191).
[0321] The analysis of pbp1a sequences from S. pneumoniae strains
with various levels of penicillin resistance from public databases
and from the database described in Example 18 allowed the
identification of amino acid substitutions Ile-459 to Met and
Ser-462 to Ala that occur in isolates with high-level penicillin
resistance (MICs.gtoreq.1 .mu.g/ml), and amino acid substitutions
Ser-575 to Thr, Gln-576 to Gly and Phe-577 to Tyr that are common
to all penicillin-resistant isolates with MICs.gtoreq.0.25
.mu.g/ml. As shown in Annex XXXI, PCR primer pair SEQ ID NOs. 1130
and 1131 were designed to detect high-level penicillin resistance
(MICs.gtoreq.1 .mu.g/ml), whereas PCR primer pair SEQ ID NOs. 1129
and 1131 were designed to detect intermediate- and high-level
penicillin resistance (MICs.gtoreq.0.25 .mu.g/ml).
[0322] The analysis of hexA sequences from the publicly available
hexA sequence and from the database described in Example 19 allowed
the design of an internal probe specific to S. pneumoniae (SEQ ID
NO. 1180) (Annex XLII). The range of mismatches between the S.
pneumoniae-specific 241-bp amplicon was from 2 to 5, in the middle
of the 19-bp probe. The analysis of pbp1a sequences from public
databases and from the database described in Example 18 allowed the
design of five internal probes containing all possible mutations to
detect the high-level penicillin resistance 383-bp amplicon (SEQ ID
NOs. 1197, 1217-1220). Alternatively, two other internal probes
(SEQ ID NOs. 2024-2025) can also be used to detect the high-level
penicillin resistance 383-bp amplicon. Five internal probes
containing all possible mutations to detect the 157-bp amplicon
which includes intermediate- and high-level penicillin resistance
were also designed (SEQ ID NOs. 1094, 1192-1193, 1214 and 1216).
Design and synthesis of primers and probes, and detection of the
probe hybridization were performed as described in Example 7. Annex
XXXI illustrates one of the internal probe for detection of the
high-level penicillin resistance 383-bp amplicon (SEQ ID NO. 1197)
and one of the internal probe for detection of the intermediate-
and high-level penicillin resistance 157-bp amplicon (SEQ ID NO.
1193).
[0323] PCR amplification. For all bacterial species, amplification
was performed from purified genomic DNA using a PTC-200
thermocycler (MJ Research). 1 .mu.l of genomic DNA at 0.1 ng/.mu.l,
or 1 .mu.l of a bacterial lysate, was transferred to a 19 .mu.l PCR
mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (H
9.0), 0.1% Triton X-100, 2.5 mM MgCl.sub.2, 0.1 .mu.M (each) of the
S. pneumoniae-specific primers SEQ ID NO. 1179 and SEQ ID NO. 1181,
0.2 .mu.M of primer SEQ ID NO. 1129, 0.7 .mu.M of primer SEQ ID NO.
1131, and 0.6 .mu.M of primer SEQ ID NO. 1130, 0.05 mM bovine serum
albumin (BSA), and 0.5 U Taq polymerase (Promega) coupled with
TaqStart.TM. antibody. In order to generate Digoxigenin
(DIG)-labeled amplicons for capture probe hybridization,
0.1.times.PCR DIG labeling four deoxynucleoside triphosphates mix
(Boehringer Mannheim GmbH) was used for amplification.
[0324] For determination of the sensitivity of the PCR assays,
10-fold dilutions of purified genomic DNA were used to determine
the minimal number of genome copies which can be detected.
[0325] Capture probe hybridization. The DIG-labeled amplicons were
hybridized to the capture probes bound to 96-well plates. The
plates were incubated with anti-DIG-alkaline phosphatase and the
chemiluminescence was measured by using a luminometer (MLX, Dynex
Technologies Inc.) after incubation with CSPD and recorded as
Relative Light Unit (RLU). The RLU ratio of tested sample with and
without captures probes was then calculated. A ratio 2.0 was
defined as a positive hybridization signal. All reactions were
performed in duplicate.
Results
[0326] Amplifications with the multiplex PCR assay. The specificity
of the assay was assessed by performing 40-cycle PCR amplifications
with the panel of gram-positive (67 species from 12 genera) and
gram-negative (33 species from 17 genera) bacterial species listed
in Table 13. All bacterial species tested other than S. pneumoniae
were negative except S. mitis and S. oralis. Ubiquity tests were
performed using a collection of 98 S. pneumoniae strains including
high-level penicillin resistance (n=53), intermediate resistance
(n=12) and sensitive (n=33) strains. There was a perfect
correlation between PCR and standard susceptibility testing for 33
penicillin-sensitive isolates. Among 12 S. pneumoniae isolates with
intermediate penicillin resistance based on susceptibility testing,
11 had intermediate resistance based on PCR, but one S. pneumoniae
isolate with penicillin MIC of 0.25 .mu.g/ml showed a high-level
penicillin resistance based on genotyping. Among 53 isolates with
high-level penicillin resistance based on susceptibility testing,
51 had high-level penicillin resistance based on PCR but two
isolates with penicillin MIC>1 .mu.g/ml showed an intermediate
penicillin resistance based on genotyping. In general, there was a
good correlation between the genotype and classical culture method
for bacterial identification and susceptibility testing.
[0327] The sensitivity of the S. pneumoniae-specific assay with
40-cycle PCR protocols was determined by using purified genomic DNA
from 9 isolates of S. pneumoniae. The detection limit was around 10
copies of genomic DNA for all of them.
[0328] Post-PCR hybridization with internal probes. The specificity
of the multiplex PCR assay coupled with capture-probe hybridization
was tested with 98 strains of S. pneumoniae, 16 strains of S. mitis
and 3 strains of S. oralis. The internal probe specific to S.
pneumoniae (SEQ ID NO. 1180) detected all 98 S. pneumoniae strains
but did not hybridize to the S. mitis and S. oralis amplicons. The
five internal probes specific to the high-level resistance amplicon
(SEQ ID NOs. 1197, 1217-1220) detected all amplification patterns
corresponding to high-level resistance. The two S. pneumoniae
strains with penicillin MIC>1 .mu.g/ml that showed an
intermediate penicillin resistance based on PCR amplification were
also intermediate resistance based on probe hybridization.
Similarly, among 12 strains with intermediate-penicillin resistance
based on susceptibility testing, 11 showed intermediate-penicillin
resistance based on hybridization with the five internal probes
specific to the intermediate and high-level resistance amplicon
(SEQ ID NOs. 1094, 1192-1193, 1214 and 1216). The strain described
above having a penicillin MIC of 0.25 .mu.g/ml which was high-level
penicillin resistance based on PCR amplification was also
high-level resistance based on probe hybridization. In summary, the
combination of the multiplex PCR and hybridization assays results
in a highly specific test for the detection of penicillin-resistant
Streptococcus pneumoniae.
Assay II:
[0329] Bacterial strains. The specificity of the multiplex PCR
assay was verified by using the same strains as those used for the
development of Assay I. The penicillin MICs (minimal inhibitory
concentrations) were measured by the broth dilution method
according to the recommended protocol of NCCLS.
[0330] PCR primers and internal probes. The analysis of pbp1a
sequences from S. pneumoniae strains with various levels of
penicillin resistance from public databases and from the database
described in Example 18 allowed the design of two primers located
in the constant region of pbp1a. PCR primer pair (SEQ ID NOs. 2015
and 2016) was designed to amplify a 888-bp variable region of pbp1a
from all S. pneumoniae strains. A series of internal probes were
designed for identification of the pbp1a mutations associated with
penicillin resistance in S. pneumoniae. For detection of high-level
penicillin resistance (MICs.gtoreq.1 .mu.g/ml), three internal
probes were designed (SEQ ID NOs. 2017-2019). Alternatively, ten
other internal probes were designed that can also be used for
detection of high-level resistance within the 888-bp pbp1a
amplicon: (1) three internal probes for identification of the amino
acid substitutions Thr-371 to Ser or Ala within the motif S370TMK
(SEQ ID NOs. 2031-2033); (2) two internal probes for detection of
the amino acid substitutions Ile-459 to Met and Ser-462 to Ala near
the motif S428RN (SEQ ID NOs. 1135 and 2026); (3) two internal
probes for identification of the amino acid substitutions Asn-443
to Asp (SEQ ID NOs. 1134 and 2027); and (4) three internal probes
for detection of all sequence variations within another region (SEQ
ID NOs. 2028-2030). For detection of high-level and intermediate
penicillin resistance (MICs 0.25 .mu.g/ml), four internal probes
were designed (SEQ ID NOs. 2020-2023). Alternatively, six other
internal probes were designed for detection of the four consecutive
amino acid substitutions T574SQF to A574TGY near the motif K557TG
(SEQ ID NOs. 2034-2039) that can also be used for detection of
intermediate- and high-level resistance within the 888-bp pbp1a
amplicon.
[0331] PCR amplification. For all bacterial species, amplification
was performed from purified genomic DNA using a PTC-200
thermocycler (MJ Research). 1 .mu.l of genomic DNA at 0.1 ng/.mu.l,
or 1 .mu.l of a bacterial lysate, was transferred to a 19 .mu.l PCR
mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH
9.0), 0.1% Triton X-100, 2.5 mM MgCl.sub.2, 0.08 .mu.M (each) of
the S. pneumoniae-specific primers SEQ ID NO. 1179 and SEQ ID NO.
1181, 0.4 .mu.M of the pbp1a-specific primer SEQ ID NO. 2015, 1.2
.mu.M of pbp1a-specific primer SEQ ID NO. 2016, 0.05 mM bovine
serum albumin (BSA), and 0.5 U Taq polymerase (Promega) coupled
with TaqStart.TM. antibody. In order to generate Digoxigenin
(DIG)-labeled amplicons for capture probe hybridization,
0.1.times.PCR DIG labeling four deoxynucleoside triphosphates mix
(Boehringer Mannheim GmbH) was used for amplification.
[0332] For determination of the sensitivities of the PCR assays,
10-fold dilutions of purified genomic DNA were used to determine
the minimal number of genome copies which can be detected.
[0333] Capture probe hybridization. The DIG-labeled amplicons were
hybridized to the capture probes bound to 96-well plates as
described for Assay I.
Results
[0334] Amplifications with the multiplex PCR assay. The specificity
of the assay was assessed by performing 40-cycle PCR amplifications
with the panel of gram-positive (67 species from 12 genera) and
gram-negative (33 species from 17 genera) bacterial species listed
in Table 13. All bacterial species tested other than S. pneumoniae
were negative except S. mitis and S. oralis. Ubiquity tests were
performed using a collection of 98 S. pneumoniae strains including
high-level penicillin resistance (n=53), intermediate resistance
(n=12) and sensitive (n=33) strains. All the above S. pneumoniae
strains produced the 888-bp amplicon corresponding to pbp1a and the
241-bp fragment corresponding to hexA.
[0335] The sensitivity of the S. pneumoniae-specific assay with
40-cycle PCR protocols was determined by using purified genomic DNA
from 9 isolates of S. pneumoniae. The detection limit was around 10
copies of genomic DNA for all of them.
[0336] Post-PCR hybridization with internal probes. The specificity
of the multiplex PCR assay coupled with capture-probe hybridization
was tested with 98 strains of S. pneumoniae, 16 strains of S. mitis
and 3 strains of S. oralis. The internal probe specific to S.
pneumoniae (SEQ ID NO. 1180) detected all 98 S. pneumoniae strains
but did not hybridize to the S. mills and S. oralis amplicons. The
three internal probes (SEQ ID NOs 2017-2019) specific to high-level
resistance detected all the 43 strains with high-level penicillin
resistance based on susceptibility testing. Among 12 isolates with
intermediate-penicillin resistance based on susceptibility testing,
11 showed intermediate-penicillin resistance based on hybridization
with 4 internal probes (SEQ ID NOs. 2020-2023) and one strain
having penicillin MIC of 0.25 .mu.g/ml was misclassified as
high-level penicillin resistance. In summary, the combination of
the multiplex PCR and hybridization assays results in a highly
specific test for the detection of penicillin-resistant
Streptococcus pneumoniae.
Example 21
Sequencing of the Vancomycin Resistance vanA, vanC1, vanC2 and
vanC3 Genes
[0337] The publicly available sequences of the vanH-vanA-vanX-vanY
locus of transposon Tnl546 from E. faecalis, vanC1 sequence from
one strain of E. gallinarum, vanC2 and vanC3 sequences from a
variety of E. casseliflavus and E. flavescens strains,
respectively, allowed the design of PCR primers able to amplify the
vanA, vanC1, vanC2 and vanC3 sequences of several Enterococcus
species. Using primer pairs van6877 and van9106 (SEQ ID NOs. 1150
and 1155), vanC1-122 and vanC1-1315 (SEQ ID NOs. 1110 and 1109),
and vanC2C3-1 and vanC2C3-1064 (SEQ ID NOs. 1108 and 1107), it was
possible to amplify and determine vanA sequences SEQ ID NOs.
1049-1057, vanC1 sequences SEQ ID NOs. 1058-1059, vanC2 sequences
SEQ ID NOs. 1060-1063 and vanC3 sequences SEQ ID NOs. 1064-1066,
respectively. Four other PCR primers (SEQ ID NOs. 1151-1154) were
also designed and used to complete the sequencing of vanA
amplification products.
Example 22
Development of a PCR Assay for the Detection and Identification of
Enterococci at Genus and Species Levels and its Associated
Resistance Genes vanA and vanB
[0338] The comparison of vanA and vanB sequences revealed conserved
regions allowing the design of PCR primers specific to both vanA
and vanB sequences (Annex XXXVIII). The PCR primer pair vanAB459
and vanAB830R (SEQ ID NOs. 1112 and 1111) was used in multiplex
with the Enterococcus-specific primers Encg313dF and Encg599c (SEQ
ID NOs. 1137 and 1136) described in Example 11. Sequence alignment
analysis of vanA and vanB sequences revealed regions suitable for
the design of internal probes specific to vanA (SEQ ID NO. 1170)
and vanB (SEQ ID NO. 1171). PCR amplification and agarose gel
electrophoresis of the amplified products were performed as
described in Example 11. The optimal cycling conditions for maximum
sensitivity and specificity were found to be 3 min. at 94.degree.
C., followed by forty cycles of two steps consisting of 1 second at
95.degree. C. and 30 seconds at 62.degree. C., plus a terminal
extension at 72.degree. C. for 2 minutes. The specificity of the
multiplex assay with 40-cycle PCR was verified by using 0.1
nanogram of purified genomic DNA from a panel of bacteria listed in
Table 10. The sensitivity of the multiplex assay with 40-cycle PCR
was verified with three strains of E. casseliflavus, eight strains
of E. gallinarum, two strains of E. flavescens, two
vancomycin-resistant strains of E. faecalis and one
vancomycin-sensitive strain of E. faecalis, three
vancomycin-resistant strains of E. faecium, one
vancomycin-sensitive strain of E. faecium and one strain of each of
the other enterococcal species listed in Table 10. The detection
limit was 1 to 10 copies of genomic DNA, depending on the
enterococcal species tested. The vanA- and vanB-specific internal
probes (SEQ ID NOs. 1170 and 1171), as well as the E. faecalis- and
E. faecium-specific internal probes (SEQ ID NOs. 1174 and 602) and
the internal probe specific to the group including E.
casseliflavus, E. gallinarum and E. flavescens (SEQ ID NO. 1122)
described in Example 11, were able to recognize
vancomycin-resistant enterococcal species with high sensitivity,
specificity and ubiquity showing a perfect correlation between the
genotypic and phenotypic analysis.
[0339] The format of the assay is not limited to the one described
above. A person skilled in the art could adapt the assay for
different formats such as PCR with real-time detection using
molecular beacon probes. Molecular beacon probes designed to be
used in this assay include, but are not limited to, SEQ ID NO. 1236
for the detection of E. faecalis, SEQ ID NO. 1235 for the detection
of E. faecium, SEQ ID NO. 1240 for the detection of vanA, and SEQ
ID NO. 1241 for the detection of vanB.
Example 23
Development of a Multiplex PCR Assay for Detection and
Identification of Vancomycin-Resistant Organisms Including
Enterococcus faecalis, Enterococcus faecium and the Group Including
Enterococcus gallinarum, Enterococcus Casseliflavus, and
Enterococcus flavescens.
[0340] The vanA and vanB genes encode the major glycopeptide
resistance phenotypes in vancomycin resistant microorganisms. To
design an assay to detect vancomycin resistant microorganisms, the
nucleotide sequence of the vanA and vanB genes were analyzed. FIGS.
13 and 14 show a nucleotide sequence alignment of the vanA and vanB
genes from the bacterial strains listed in Tables 26 and 27,
respectively. Shown above the sequence alignments is a consensus
DNA sequence derived from the multiple sequences. The analysis of
vanA and vanB sequences revealed conserved regions allowing design
of PCR primer pairs (SEQ ID NOs. 1089 and 1090 and SEQ ID NOs: 1090
and 1091) specific to vanA sequences (Annex XXVIII) and PCR primer
pairs (SEQ ID NOs. 1095 and 1096 and SEQ ID NOs: 2298 and 1096)
specific to vanB sequences (Annex XXIX). Shown below the sequence
alignments in FIGS. 13 and 14 is the sequence and location of the
vanA primers SEQ ID NO: 1090 and 1091 and the vanB primers SEQ ID
NOs: 2298 and 1096, respectively. Also shown are the sequence and
location of molecular beacon probes, SEQ ID NOs: 2299, 2300,
designed for the detection of the vanA and vanB amplification
products, respectively. SEQ ID NO: 2299. The vanA molecular beacon
(SEQ ID NO: 2299) contains a 5' carboxyfluorescein (FAM)
fluorophore. The vanB molecular beacon (SEQ ID NO: 2300) contains a
sulforhodamine active ester (Texas Red) at its 5' end. Both beacons
contain the nonfluorescent quencher moiety dabcyl chloride (DABCYL)
at their 3' ends.
[0341] An internal control DNA, pERVd (SEQ ID NO: 2302) was
designed for the vanR assay. The primers of SEQ ID NO: 1090 and SEQ
ID NO: 1096 can hybridize to produce and amplification product from
the internal control DNA. A molecular beacon probe (SEQ ID NO:
2301) was designed to detect the amplification product of the
internal control DNA. SEQ ID NO: 2301 contains a 5'
tetrachlorofluorescein (TET) fluorophore and the DABCYL quencher
moiety at its 3' end.
[0342] In a first experiment, the vanA-specific PCR primer pair
(SEQ ID. NOs. 1089 and 1090) was used in multiplex with the
vanB-specific PCR primer pair described in our assigned U.S. Pat.
No. 5,994,066 (SEQ ID NOs. 1095 and 1096 in the present patent and
SEQ ID NOs. 231 and 232 in the said patent). The comparison of
vanC1, vanC2 and vanC3 sequences revealed conserved regions
allowing design of PCR primers (SEQ ID NOs. 1101 and 1102) able to
generate a 158-bp amplicon specific to the group including E.
gallinarum, E. casseliflavus and E. flavescens (Annex XXX). The
vanC-specific PCR primer pair (SEQ ID NOs. 1101 and 1102) was used
in multiplex with the E. faecalis-specific PCR primer pair
described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 40
and 41 in the said patent) and with the E. faecium-specific PCR
primer pair described in our patent publication WO98/20157 (SEQ ID
NOs. 1 and 2 in the said publication). For both multiplexes, the
optimal cycling conditions for maximum sensitivity and specificity
were found to be 3 min. at 94.degree. C., followed by forty cycles
of two steps consisting of 1 second at 95.degree. C. and 30 seconds
at 58.degree. C., plus a terminal extension at 72.degree. C. for 2
minutes. Detection of the PCR products was made by electrophoresis
in agarose gels (2%) containing 0.25 .mu.g/ml of ethidium bromide.
The vanA-specific PCR primer pair (SEQ ID NOs. 1089 and 1090), the
vanB-specific primer pair (SEQ ID NOs. 1095 and 1096) and the
vanC-specific primer pair (SEQ ID NOs. 1101 and 1102) were tested
for their specificity by using 0.1 nanogram of purified genomic DNA
from a panel of 5 vancomycin-sensitive Enterococcus species, 3
vancomycin-resistant Enterococcus species, 13 other gram-positive
bacteria and one gram-negative bacterium. Specificity tests were
performed with the E. faecium-specific PCR primer pair described in
our patent publication WO98/20157 (SEQ ID NOs. 1 and 2 in the said
publication) and with the E. faecalis-specific PCR primer pair
described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 40
and 41 in the said patent) on a panel of 37 gram-positive bacterial
species. All Enterococcus strains were amplified with high
specificity showing a perfect correlation between the genotypic and
phenotypic analysis. The sensitivity of the assays was determined
for several strains of E. gallinarum, E. casseliflavus, E.
flavescens and vancomycin-resistant E. faecalis and E. faecium.
Using each of the E. faecalis- and E. faecium-specific PCR primer
pairs as well as vanA-, vanB- and vanC-specific PCR primers used
alone or in multiplex as described above, the sensitivity ranged
from 1 to 10 copies of genomic DNA.
[0343] The format of the assay is not limited to the one described
above. A person skilled in the art could adapt the assay for
different formats such as PCR with real-time detection using
molecular beacon probes. Molecular beacon probes designed to be
used in this assay include, but are not limited to, SEQ ID NO. 1238
for the detection of E. faecalis, SEQ ID NO. 1237 for the detection
of E. faecium, SEQ ID NO. 1239 for the detection of vanA, and SEQ
ID NO. 1241 for the detection of vanB.
[0344] Another PCR assay was developed for the detection of
vancomycin-resistant E. faecium and vancomycin-resistant E.
faecalis. This assay included two multiplex: (1) the first
multiplex contained the vanA-specific primer pair (SEQ ID NOs.
1090-1091) and the vanB-specific PCR primer pair described in our
assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 1095 and 1096 in the
present patent and SEQ ID NOs. 231 and 232 in the said patent), and
(2) the second multiplex contained the E. faecalis-specific PCR
primer pair described in our assigned U.S. Pat. No. 5,994,066 (SEQ
ID NOs. 40 and 41 in the said patent) and the E. faecium-specific
PCR primer pair described in our patent publication WO98/20157 (SEQ
ID NOs. 1 and 2 in the said publication). For both multiplexes, the
optimal cycling conditions for maximum sensitivity and specificity
were found to be 3 min. at 94.degree. C., followed by forty cycles
of two steps consisting of 1 second at 95.degree. C. and 30 seconds
at 58.degree. C., plus a terminal extension at 72.degree. C. for 2
minutes. Detection of the PCR products was made by electrophoresis
in agarose gels (2%) containing 0.25 .mu.g/ml of ethidium bromide.
The two multiplexes were tested for their specificity by using 0.1
nanogram of purified genomic DNA from a panel of two
vancomycin-sensitive E. faecalis strains, two vancomycin-resistant
E. faecalis strains, two vancomycin-sensitive E. faecium strains,
two vancomycin-resistant E. faecium strains, 16 other enterococcal
species and 31 other gram-positive bacterial species. All the E.
faecium and E. faecalis strains were amplified with high
specificity showing a perfect correlation between the genotypic
analysis and the susceptibility to glycopeptide antibiotics
(vancomycin and teicoplanin). The sensitivity of the assay was
determined for two vancomycin-resistant E. faecalis strains and two
vancomycin-resistant E. faecium strains. The detection limit was 5
copies of genomic DNA for all the strains.
[0345] This multiplex PCR assay was coupled with capture-probe
hybridization. Four internal probes were designed: one specific to
the vanA amplicon (SEQ ID NO. 2292), one specific to the vanB
amplicon (SEQ ID NO. 2294), one specific to the E. faecalis
amplicon (SEQ ID NO. 2291) and one specific to the E. faecium
amplicon (SEQ ID NO. 2287). Each of the internal probes detected
their specific amplicons with high specificity and sensitivity.
The VanR Assay
Development
[0346] In a next set of experiments, a multiplex real-time PCR
reaction (the VanR assay) using the vanA primer pair SEQ ID NO:
1090 and 1091, the vanB primer pair SEQ ID NO: 1096 and 2298, the
internal control DNA SEQ ID NO: 2302, and the molecular beacon
probes SEQ ID NOs: 2299, 2300, and 2301 was performed.
[0347] For the template DNA, purified genomic DNA from vanA
resistant Enterococcus faecium or vanB resistant Enterococcus
faecalis was obtained from strains grown on blood agar plates under
standard conditions. Genomic DNA was extracted from the cultures
using the GNOME kit (QBIOgene, Carlsbad, Calif.) according to the
manufacturer's instructions. Template DNA samples were treated with
RNase prior to quantitation. Genomic DNA concentration was
determined and the quality of the genomic DNA preparations was
confirmed using conventional methods.
[0348] Lyophilized reagents listed in Table 24 were used for the
VanR assay. The lyophilized reagents were reconstituted with 225
.mu.l diluent (116 mM Tris-HCl, pH 8.3, 11.6 mM KCl, 3.48 mM
MgCl.sub.2, 5.8 mM NH.sub.2SO.sub.4) and subsequently divided into
25 .mu.l aliquots. 0.5, 2.5, 5, 10 or 20 copies of template DNA was
added to each of 5 replicate reactions.
[0349] The VanR assay PCR was run in a SMART CYCLER.TM. PCR machine
under the following conditions: 60.degree. C. for 6 sec, followed
by 95.degree. C. for 900 sec, followed by 45 cycles of 95.degree.
C. for 5 seconds, 63.degree. C. for 10 sec and 72.degree. C. for 20
sec. The fluorescent readout from the FAM channel (vanA) and the
Texas Red channel (vanB) of the reactions is shown in FIGS. 13A and
13B, respectively. The VanR assay is capable of detecting 5 copies
of vanA target DNA in a sample (FIG. 13A) and of detecting 10
copies of vanB target DNA in a sample (FIG. 13B). For each
concentration of template DNA, the number of positive assay results
out of each of the five replicates was recorded (Table 15).
Specificity
[0350] To demonstrate the specificity of the VanR assay, vanC,
vanD, vanE, and vanG resistant enterococci, other closely related
bacteria, normal and pathogenic anal or fecal flora, or human DNA
as listed in Tables 16-18 were assayed as follows: VanR assay
lyophilized reagents (Table 38) were reconstituted with 225 .mu.l
diluent and aliquoted as described above. Template DNA was prepared
using conventional methods and diluted in sample preparation buffer
to a final concentration of 0.33 ng/.mu.l. 3 .mu.l template DNA was
added to each master mix, and the reaction was carried out as
described above in the SMART CYCLER.TM. PCR machine. 41
electrophoresis loading buffer was added to the reactions upon
completion, and 15 .mu.l of each reaction was run on an agarose gel
to view the PCR amplification products, as shown in FIGS.
14-17.
[0351] To assess the specificity of the molecular beacons in the
VanR assay, PCR assays were preformed to test cross-reactivity with
amplified vanA and vanB target DNA and internal control DNA. The
reaction components listed in Table 34 were each added to premix in
Table 33. For each reaction, 25.8 .mu.l of the final mix was
combined with 3 .mu.l template DNA (either 25 copies vanA DNA or 50
copies vanB DNA) or TE buffer (1.times.), as indicated. The
reactions were performed in the SMART CYCLER.TM. PCR machine using
the VanR assay conditions as described above. Table 35 shows the
results of the experiment. Positive results were obtained in the
FAM channel in every reaction that contained vanA template DNA and
the vanA primers. Positive results were obtained in the Texas Red
channel in every reaction that contained vanB template DNA and vanB
primers. Positive results obtained in the TET channel in every
reaction that contained internal control template DNA and internal
control primers.
[0352] Negative results were obtained in each channel for every
reaction that did not contain a target DNA that could be amplified
and detected using the indicated molecular beacon probe. In other
words, the molecular beacon probes did not cross-react with
non-specific DNAs. FIGS. 18A-20B show the fluorescent readouts of
the experiments.
Validation with Clinical Samples
[0353] To validate the VanR assay, the sensitivity of the VanR
assay was performed on clinical samples. Briefly, the VanR assay
lyophilized reagents listed in Table 38 were reconstituted in 225
.mu.l diluent and aliquoted as described above. DNA isolation and
quantitation was performed using conventional techniques. The
samples were processed in a SMART CYCLER' PCR machine using the
VanR reaction conditions described above.
[0354] FIG. 21 shows the fluorescent readout in the FAM (vanA)
channel and the Texas Red channel (vanB) from a specimen that is
vanA resistant. The reaction gave a positive result in the FAM
channel but not the Texas Red channel. FIG. 22 shows the
fluorescent readout in the FAM and Texas Red channels from a
combination of a vanA resistant specimen and a vanB resistant
specimen. Positive results were obtained in both the FAM and the
Texas Red channel. FIG. 23 shows the fluorescent readout in the FAM
and Texas Red channels from a vanB positive specimen. The results
were negative in the FAM channel, and positive in the Texas Red
channel.
Sensitivity
[0355] To assess the sensitivity of the VanR assay, template DNA
isolated from enterococci from various geographic regions, and on
template DNA from vanB resistant bacterial species other than
enterococci was isolated and tested in the VanR assay as described
above. Table 36 lists the enterococcal strains from various regions
in the world tested in the assay. The table identifies the vanA and
vanB phenotype of each strain. PCR amplification products were
detected by running a sample of each reaction on an agarose gel,
shown in FIG. 24. vanA or vanB PCR amplification products were
detectable in each of the strains tested. Table 37 lists
non-enterococcal, vanB resistant strains tested in the VanR assay.
The PCR amplification products are shown in FIG. 25. The reaction
produced an amplification product of the expected size for the vanB
amplicon.
[0356] These results demonstrate that the VanR assay is sensitive
and specific.
Example 24
Universal Amplification Involving the EF-G (fusA) Subdivision of
tuf Sequences
[0357] As shown in FIG. 3, primers SEQ ID NOs. 1228 and 1229 were
designed to amplify the region between the end of fusA and the
beginning of tuf genes in the str operon. Genomic DNAs from a panel
of 35 strains were tested for PCR amplification with those primers.
In the initial experiment, the following strains showed a positive
result: Abiotrophia adiacens ATCC 49175, Abiotrophia defectiva ATCC
49176, Bacillus subtilis ATCC 27370, Clostridium difficile ATCC
9689, Enterococcus avium ATCC 14025, Enterococcus casseliflavus
ATCC 25788, Enterococcus cecorum ATCC 43198, Enterococcus faecalis
ATCC 29212, Enterococcus faecium ATCC 19434, Enterococcus
flavescens ATCC 49996, Enterococcus gallinarum ATCC 49573,
Enterococcus solitarius ATCC 49428, Escherichia coli ATCC 11775,
Haemophilus influenzae ATCC 9006, Lactobacillus acidophilus ATCC
4356, Peptococcus niger ATCC 27731, Proteus mirabilis ATCC 25933,
Staphylococcus aureus ATCC 43300, Staphylococcus auricularis ATCC
33753, Staphylococcus capitis ATCC 27840, Staphylococcus epidemidis
ATCC 14990, Staphylococcus haemolyticus ATCC 29970, Staphylococcus
hominis ATCC 27844, Staphylococcus lugdunensis ATCC 43809,
Staphylococcus saprophyticus ATCC 15305, Staphylococcus simulans
ATCC 27848, and Staphylococcus warneri ATCC 27836. This primer pair
could amplify additional bacterial species; however, there was no
amplification for some species, suggesting that the PCR cycling
conditions could be optimized or the primers modified. For example,
SEQ ID NO. 1227 was designed to amplify a broader range of
species.
[0358] In addition to other possible primer combinations to amplify
the region covering fusA and tuf, FIG. 3 illustrates the positions
of amplification primers SEQ ID NOs. 1221-1227 which could be used
for universal amplification of fusA segments. All of the above
mentioned primers (SEQ ID NOs. 1221-1229) could be useful for the
universal and/or the specific detection of bacteria.
[0359] Moreover, different combinations of primers SEQ ID NOs.
1221-1229, sometimes in combination with tuf sequencing primer SEQ
ID NO. 697, were used to sequence portions of the str operon,
including the intergenic region. In this manner, the following
sequences were generated: SEQ ID NOs. 1518-1526, 1578-1580,
1786-1821, 1822-1834, 1838-1843, 2184, 2187, 2188, 2214-2249, and
2255-2269.
Example 25
DNA Fragment Isolation from Staphylococcus saprophyticus by
Arbitrarily Primed PCR
[0360] DNA sequences of unknown coding potential for the
species-specific detection and identification of Staphylococcus
saprophyticus were obtained by the method of arbitrarily primed PCR
(AP-PCR).
[0361] AP-PCR is a method which can be used to generate specific
DNA probes for microorganisms (Fani et al., 1993, Molecular Ecology
2:243-250). A description of the AP-PCR protocol used to isolate a
species-specific genomic DNA fragment from Staphylococcus
saprophyticus follows. Twenty different oligonucleotide primers of
10 nucleotides in length (all included in the AP-PCR kit OPAD
(Operon Technologies, Inc., Alameda, Calif.)) were tested
systematically with DNAs from 5 bacterial strains of Staphylococcus
saprophyticus as well as with bacterial strains of 27 other
staphylococcal (non-S. saprophyticus) species. For all bacterial
species, amplification was performed directly from one .mu.L (0.1
ng/.mu.L) of purified genomic DNA. The 25 .mu.L PCR reaction
mixture contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton
X-100, 2.5 mM MgCl.sub.2, 1.2 .mu.M of only one of the 20 different
AP-PCR primers OPAD, 200 .mu.M of each of the four dNTPs, 0.5 U of
Taq DNA polymerase (Promega Corp., Madison, Wis.) coupled with
TaqStart.TM. antibody (Clontech Laboratories Inc., Palo Alto,
Calif.). PCR reactions were subjected to cycling using a MJ
Research PTC-200 thermal cycler as follows: 3 min at 96.degree. C.
followed by 42 cycles of 1 min at 94.degree. C. for the
denaturation step, 1 min at 31.degree. C. for the annealing step
and 2 min at 72.degree. C. for the extension step. A final
extension step of 7 min at 72.degree. C. followed the 42 cycles to
ensure complete extension of PCR products. Subsequently, twenty
microliters of the PCR-amplified mixture were resolved by
electrophoresis on a 1.5% agarose gel containing 0.25 .mu.g/ml of
ethidium bromide. The size of the amplification products was
estimated by comparison with a 50-bp molecular weight ladder.
[0362] Amplification patterns specific for Staphylococcus
saprophyticus were observed with the AP-PCR primer OPAD-16
(sequence: 5'-AACGGGCGTC-3'). Amplification with this primer
consistently showed a band corresponding to a DNA fragment of
approximately 380 by for all Staphylococcus saprophyticus strains
tested but not for any of the other staphylococcal species
tested.
[0363] The band corresponding to the 380 by amplicon, specific and
ubiquitous for S. saprophyticus based on AP-PCR, was excised from
the agarose gel and purified using the QIAquick.TM. gel extraction
kit (QIAGEN Inc.). The gel-purified DNA fragment was cloned into
the T/A cloning site of the pCR 2.1.TM. plasmid vector (Invitrogen
Inc.) using T4 DNA ligase (New England BioLabs). Recombinant
plasmids were transformed into E. coli DH5.alpha. competent cells
using standard procedures. All reactions were performed according
to the manufacturer's instructions. Plasmid DNA isolation was done
by the method of Birnboim and Doly (Nucleic Acid Res., 1979,
7:1513-1523) for small-scale preparations. All plasmid DNA
preparations were digested with the EcoRI restriction endonuclease
to ensure the presence of the approximately 380 by AP-PCR insert
into the plasmid. Subsequently, a large-scale and highly purified
plasmid DNA preparation was performed from two selected clones
shown to carry the AP-PCR insert by using the QIAGEN plasmid
purification kit (midi format). These large-scale plasmid
preparations were used for automated DNA sequencing.
[0364] The 380 by nucleotide sequence was determined for three
strains of S. saprophyticus (SEQ ID NOs. 74, 1093, and 1198). Both
strands of the AP-PCR insert from the two selected clones were
sequenced by the dideoxynucleotide chain termination sequencing
method with SP6 and T7 sequencing primers by using the Applied
Biosystems automated DNA sequencer (model 373A) with their
PRISM.TM. Sequenase.RTM. Terminator Double-stranded DNA Sequencing
Kit (Applied Biosystems, Foster City, Calif.).
[0365] Optimal species-specific amplification primers (SEQ ID NOs.
1208 and 1209) have been selected from the sequenced AP-PCR
Staphylococcus saprophyticus DNA fragments with the help of the
primer analysis software Oligo.TM. 5.0 (National BioSciences Inc).
The selected primers were tested in PCR assays to verify their
specificity and ubiquity. Data obtained with DNA preparations from
reference ATCC strains of 49 gram-positive and 31 gram-negative
bacterial species, including 28 different staphylococcal species,
indicate that the selected primer pairs are specific for
Staphylococcus saprophyticus since no amplification signal has been
observed with DNAs from the other staphylococcal or bacterial
species tested. This assay was able to amplify efficiently DNA from
all 60 strains of S. saprophyticus from various origins tested. The
sensitivity level achieved for three S. saprophyticus reference
ATCC strains was around 6 genome copies.
Example 26
Sequencing of Prokaryotic tuf Gene Fragments
[0366] The comparison of publicly available tuf sequences from a
variety of bacterial species revealed conserved regions, allowing
the design of PCR primers able to amplify tuf sequences from a wide
range of bacterial species. Using primer pair SEQ ID NOs. 664 and
697, it was possible to amplify and determine tuf sequences SEQ ID
NOs.: 1-73, 75-241, 607-618, 621, 662, 675, 717-736, 868-888, 932,
967-989, 992, 1002, 1572-1575, 1662-1663, 1715-1733, 1835-1837,
1877-1878, 1880-1881, 2183, 2185, 2200, 2201, and 2270-2272.
Example 27
Sequencing of Procaryotic recA Gene Fragments
[0367] The comparison of publicly available recA sequences from a
variety of bacterial species revealed conserved regions, allowing
the design of PCR primers able to amplify recA sequences from a
wide range of bacterial species. Using primer pairs SEQ ID NOs.
921-922 and 1605-1606, it was possible to amplify and determine
recA sequences SEQ ID NOs.: 990-991, 1003, 1288-1289, 1714,
1756-1763, 1866-1873 and 2202-2212.
Example 28
Specific Detection and Identification of Escherichia coli/Shigella
Sp. Using tuf Sequences
[0368] The analysis of tuf sequences from a variety of bacterial
species allowed the selection of PCR primers (SEQ ID NOs. 1661 and
1665) and of an internal probe (SEQ ID NO. 2168) specific to
Escherichia coli/Shigella sp. The strategy used to design the PCR
primers was based on the analysis of a multiple sequence alignment
of various tuf sequences. The multiple sequence alignment included
the tuf sequences of Escherichia coli/Shigella sp. as well as tuf
sequences from other species and bacterial genera, especially
representatives of closely related species. A careful analysis of
this alignment allowed the selection of oligonucleotide sequences
which are conserved within the target species but which
discriminate sequences from other species, especially from the
closely related species, thereby permitting the species-specific
and ubiquitous detection and identification of the target bacterial
species.
[0369] The chosen primer pair, oligos SEQ ID NOs. 1661 and 1665,
gives an amplification product of 219 bp. Standard PCR was carried
out using 0.4 .mu.M of each primer, 2.5 mM MgCl.sub.2, BSA 0.05 mM,
50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, dNTPs 0.2 mM
(Pharmacia), 0.5 U Taq DNA polymerase (Promega) coupled with
TaqStart.TM. antibody (Clontech Laboratories Inc.), 1 .mu.l of
genomic DNA sample in a final volume of 20 .mu.l using a PTC-200
thermocycler (MJ Research). The optimal cycling conditions for
maximum sensitivity and specificity were 3 minutes at 95.degree. C.
for initial denaturation, then forty cycles of two steps consisting
of 1 second at 95.degree. C. and 30 seconds at 60.degree. C.,
followed by terminal extension at 72.degree. C. for 2 minutes.
Detection of the PCR products was made by electrophoresis in
agarose gels (2%) containing 0.25 .mu.g/ml of ethidium bromide.
Visualization of the PCR products was made under UV at 254 nm.
[0370] Specificity of the assay was tested by adding to the PCR
reactions 0.1 ng of genomic DNA from each of the following
bacterial species: Escherichia coli (7 strains), Shigella sonnei,
Shigella flexneri, Shigella dysenteriae, Salmonella typhimyurium,
Salmonella typhi, Salmonella enteritidis, Tatumella ptyseos,
Klebsiella pneumoniae (2 strains), Enterobacter aerogenes,
Citrobacter farmeri, Campylobacter jejuni, Serratia marcescens.
Amplification was observed only for the Escherichia coli and
Shigella sp. strains listed and Escherichia fergusonii. The
sensitivity of the assay with 40-cycle PCR was verified with one
strain of E. coli and three strains of Shigella sp. The detection
limit for E. coli and Shigella sp. was 1 to 10 copies of genomic
DNA, depending on the strains tested.
Example 29
Specific Detection and Identification of Klebsiella pneumoniae
Using atpD Sequences
[0371] The analysis of atpD sequences from a variety of bacterial
species allowed the selection of PCR primers specific to K.
pneumoniae. The primer design strategy is similar to the strategy
described in Example 28 except that atpD sequences were used in the
alignment.
[0372] Two K. pneumoniae-specific primers were selected, (SEQ ID
NOs. 1331 and 1332) which give an amplification product of 115 bp.
Standard PCR was carried out on PTC-200 thermocyclers (MJ Research)
using 0.4 .mu.M of each primer as described in Example 28. The
optimal cycling conditions for maximum sensitivity and specificity
were as follow: three minutes at 95.degree. C. for initial
denaturation, then forty cycles of two steps consisting of 1 second
at 95.degree. C. and 30 seconds at 55.degree. C., followed by
terminal extension at 72.degree. C. for 2 minutes.
[0373] Specificity of the assay was tested by adding to the PCR
reactions 0.1 ng of genomic DNA from each of the following
bacterial species: Klebsiella pneumoniae (2 strains), Klebsiella
ornitholytica, Klebsiella oxytoca (2 strains), Klebsiella
planticola, Klebsiella terrigena, Citrobacter freundii, Escherichia
coli, Salmonella cholerasuis typhi, Serratia marcescens,
Enterobacter aerogenes, Proteus vulgaris, Kluyvera ascorbata,
Kluyvera georgiana, Kluyvera cryocrescens and Yersinia
enterolitica. Amplification was detected for the two K. pneumoniae
strains, K. planticola, K. terrigena and the three Kluyvera species
tested. Analysis of the multiple alignment sequence of the atpD
gene allowed the design of an internal probe SEQ ID NO. 2167 which
can discriminate Klebsiella pneumoniae from other Klebsiella sp.
and Kluyvera sp. The sensitivity of the assay with 40-cycle PCR was
verified with one strain of K. pneumoniae. The detection limit for
K. pneumoniae was around 10 copies of genomic DNA.
Example 30
Specific Detection and Identification of Acinetobacter baumannii
Using atpD Sequences
[0374] The analysis of atpD sequences from a variety of bacterial
species allowed the selection of PCR primers specific to
Acinetobacter baumannii. The primer design strategy is similar to
the strategy described in Example 28.
[0375] Two A. baumannii-specific primers were selected, SEQ ID NOs.
1690 and 1691, which give an amplification product of 233 bp.
Standard PCR was carried out on PTC-200 thermocyclers (MJ Research)
using 0.4 .mu.M of each primer as described in Example 28. The
optimal cycling conditions for maximum sensitivity and specificity
were as follow: three minutes at 95.degree. C. for initial
denaturation, then forty cycles of two steps consisting of 1 second
at 95.degree. C. and 30 seconds at 60.degree. C., followed by
terminal extension at 72.degree. C. for 2 minutes.
[0376] Specificity of the assay was tested by adding to the PCR
reactions 0.1 ng of genomic DNA from each of the following
bacterial species: Acinetobacter baumannii (3 strains),
Acinetobacter anitratus, Acinetobacter lwoffi, Serratia marcescens,
Enterobacter cloacae, Enterococcus faecalis, Pseudomonas
aeruginosa, Psychrobacter phenylpyruvicus, Neisseria gonorrheoae,
Haemophilus haemoliticus, Yersinia enterolitica, Proteus vulgaris,
Eikenella corrodens, Escherichia coli. Amplification was detected
only for A. baumannii, A. anitratus and A. lwoffi. The sensitivity
of the assay with 40-cycle PCR was verified with two strains of A.
baumannii. The detection limit for the two A. baumannii strains
tested was 5 copies of genomic DNA. Analysis of the multiple
alignment sequence of the atpD gene allowed the design of a A.
baumannii-specific internal probe (SEQ ID NO. 2169).
Example 31
Specific Detection and Identification of Neisseria gonorrhoeae
Using tuf Sequences
[0377] The analysis of tuf sequences from a variety of bacterial
species allowed the selection of PCR primers specific to Neisseria
gonorrhoeae. The primer design strategy is similar to the strategy
described in Example 28.
[0378] Two N. gonorrhoeae-specific primers were selected, SEQ ID
NOs. 551 and 552, which give an amplification product of 139 bp.
PCR amplification was carried out on PTC-200 thermocyclers (MJ
Research) using 0.4 .mu.M of each primer as described in Example
28. The optimal cycling conditions for maximum sensitivity and
specificity were as follow: three minutes at 95.degree. C. for
initial denaturation, then forty cycles of two steps consisting of
1 second at 95.degree. C. and 30 seconds at 65.degree. C., followed
by terminal extension at 72.degree. C. for 2 minutes.
[0379] Specificity of the assay was tested by adding into the PCR
reactions, 0.1 ng of genomic DNA from each of the following
bacterial species: Neisseria gonorrhoeae (19 strains), Neisseria
meningitidis (2 strains), Neisseria lactamica, Neisseria
flavescens, Neisseria animalis, Neisseria canis, Neisseria
cuniculi, Neisseria elongata, Neisseria mucosa, Neisseria
polysaccharea, Neisseria sicca, Neisseria subflava, Neisseria
weaveri. Amplification was detected only for N. gonorrhoeae, N.
sicca and N. polysaccharea. The sensitivity of the assay with
40-cycle PCR was verified with two strains of N. gonorrhoeae. The
detection limit for the N. gonorrhoeae strains tested was 5 copies
of genomic DNA. Analysis of the multiple alignment sequence of the
tuf gene allowed the design of an internal probe, SEQ ID NO. 2166,
which can discriminate N. gonorrhoeae from N. sicca and N.
polysaccharea.
Example 32
Sequencing of Bacterial gyrA and parC Gene Fragments. SEQUENCING of
Bacterial gyrA and parC Fragments
[0380] One of the major mechanism of resistance to quinolone in
various bacterial species is mediated by target changes (DNA gyrase
and/or topoisomerase IV). These enzymes control DNA topology and
are vital for chromosome function and replication. Each of these
enzymes is a tetramer composed of two subunits: GyrA and GyrB
forming A.sub.2B.sub.2 complex in DNA gyrase; and ParC and ParE
forming and C.sub.2E.sub.2 complex in DNA topoisomerase IV. It has
been shown that they are hotspots, called the
quinolone-resistance-determining region (QRDR) for mutations within
gyrA that encodes for the GyrA subunit of DNA gyrase and within
parC that encodes the parC subunit of topoisomerase IV.
[0381] In order to generate a database for gyrA and parC sequences
that can be used for design of primers and/or probes for the
specific detection of quinolone resistance in various bacterial
species, gyrA and parC DNA fragments selected from public database
(GenBanK and EMBL) from a variety of bacterial species were used to
design oligonucleotide primers.
[0382] Using primer pair SEQ ID NOs. 1297 and 1298, it was possible
to amplify and determine gyrA sequences from Klebsiella oxytoca
(SEQ ID NO. 1764), Klebsiella pneumoniae subsp. ozaneae (SEQ ID NO.
1765), Klebsiella planticola (SEQ ID NO. 1766), Klebsiella
pneumoniae (SEQ ID NO. 1767), Klebsiella pneumoniae subsp.
pneumoniae (two strains) (SEQ ID NOs. 1768-1769), Klebsiella
pneumoniae subsp. rhinoscleromatis (SEQ ID NO. 1770), Klebsiella
terrigena (SEQ ID NO. 1771), Kluyvera ascorbata (SEQ ID NO. 2013),
Kluyvera georgiana (SEQ ID NO. 2014) and Escherichia coli (4
strains) (SEQ ID NOs. 2277-2280). Using primer pair SEQ ID NOs.
1291 and 1292, it was possible to amplify and determine gyrA
sequences from Legionella pneumophila subsp. pneumophila (SEQ ID
NO. 1772), Proteus mirabilis (SEQ ID NO. 1773), Providencia
rettgeri (SEQ ID NO. 1774), Proteus vulgaris (SEQ ID NO. 1775) and
Yersinia enterolitica (SEQ ID NO. 1776). Using primer pair SEQ ID
NOs. 1340 and 1341, it was possible to amplify and determine gyrA
sequence from Staphylococcus aureus (SEQ ID NO. 1255).
[0383] Using primers SEQ ID NOs. 1318 and 1319, it was possible to
amplify and determine parC sequences from K oxytoca (two strains)
(SEQ ID NOs. 1777-1778), Klebsiella pneumoniae subsp. ozaenae (SEQ
ID NO. 1779), Klebsiella planticola (SEQ ID NO. 1780), Klebsiella
pneumoniae (SEQ ID NO. 1781), Klebsiella pneumoniae subsp.
pneumoniae (two strains) (SEQ ID NOs. 1782-1783), Klebsiella
pneumoniae subsp. rhinoscleromatis (SEQ ID NO. 1784) and Klebsiella
terrigena (SEQ ID NO. 1785).
Example 33
Development of a PCR Assay for the Specific Detection and
Identification of Staphylococcus aureus and its Quinolone
Resistance Genes gyrA and parC
[0384] The analysis of gyrA and parC sequences from a variety of
bacterial species revealed conserved regions allowing the design of
PCR primers specific to the quinolone-resistance-determining region
(QRDR) of gyrA and parC from Staphylococcus aureus. PCR primer pair
SEQ ID NOs. 1340 and 1341 was designed to amplify the gyrA sequence
of S. aureus, whereas PCR primer pair SEQ ID NOs. 1342 and 1343 was
designed to amplify S. aureus parC. The comparison of gyrA and parC
sequences from S. aureus strains with various levels of quinolone
resistance allowed the identification of amino acid substitutions
Ser-84 to Leu, Glu-88 to Gly or Lys in the GyrA subunit of DNA
gyrase encoded by gyrA and amino acid changes Ser-80 to Phe or Tyr
and Ala-116 to Glu in the ParC subunit of topoisomerase IV encoded
by parC. These amino acid substitutions in GyrA and ParC subunits
occur in isolates with intermediate- or high-level quinolone
resistance. Internal probes for the specific detection of wild-type
S. aureus gyrA (SEQ ID NO. 1940) and wild-type S. aureus parC (SEQ
ID NO. 1941) as well as internal probes for the specific detection
of each of the gyrA (SEQ ID NOs. 1333-1335) and parC mutations
identified in quinolone-resistant S. aureus (SEQ ID NOs. 1336-1339)
were designed.
[0385] The gyrA- and parC-specific primer pairs (SEQ ID NOs.
1340-1341 and SEQ ID NOs. 1342-1343) were used in multiplex. PCR
amplification was carried out on PTC-200 thermocyclers (MJ
Research) using 0.3, 0.3, 0.6 and 0.6 .mu.M of each primers,
respectively, as described in Example 28. The optimal cycling
conditions for maximum sensitivity and specificity were 3 minutes
at 95.degree. C. for initial denaturation, then forty cycles of two
steps consisting of 1 second at 95.degree. C. and 30 seconds at
62.degree. C., followed by terminal extension at 72.degree. C. for
2 minutes. Detection of the PCR products was made by
electrophoresis in agarose gels (2%) containing 0.25 .mu.g/ml of
ethidium bromide. The specificity of the multiplex assay with
40-cycle PCR was verified by using 0.1 ng of purified genomic DNA
from a panel of gram-positive bacteria. The list included the
following: Abiotrophia adiacens, Abiotrophia defectiva, Bacillus
cereus, Bacillus mycoides, Enterococcus faecalis (2 strains),
Enterococcus flavescens, Gemella morbillorum, Lactococcus lactis,
Listeria innocua, Listeria monocytogenes, Staphylococcus aureus (5
strains), Staphylococcus auricalis, Staphylococcus capitis subsp.
urealyticus, Staphylococcus carnosus, Staphylococcus chromogenes,
Staphylococcus epidermidis (3 strains), Staphylococcus gallinarum,
Staphylococcus haemolyticus (2 strains), Staphylococcus hominis,
Staphylococcus hominis subsp hominis, Staphylococcuslentus,
Staphylococcus lugdunensis, Staphylococcus saccharolyticus,
Staphylococcus saprophyticus (3 strains), Staphylococcus simulans,
Staphylococcus warneri, Staphylococcus xylosus, Streptococcus
agalactiae, Streptococcus pneumoniae. Strong amplification of both
gyrA and parC genes was only detected for the S. aureus strains
tested. The sensitivity of the multiplex assay with 40-cycle PCR
was verified with one quinolone-sensitive and four
quinolone-resistant strains of S. aureus. The detection limit was 2
to 10 copies of genomic DNA, depending on the strains tested.
[0386] Detection of the hybridization with the internal probes was
performed as described in Example 7. The internal probes specific
to wild-type gyrA and parC of S. aureus and to the gyrA and parC
variants of S. aureus were able to recognize two
quinolone-resistant and one quinolone-sensitive S. aureus strains
showing a perfect correlation with the susceptibility to
quinolones.
[0387] The complete assay for the specific detection of S. aureus
and its susceptibility to quinolone contains the
Staphylococcus-specific primers (SEQ ID NOs. 553 and 575) described
in Example 7 and the multiplex containing the S. aureus gyrA- and
parC-specific primer pairs (SEQ ID NOs. 1340-1341 and SEQ ID NOs.
1342-1343). Amplification is coupled with post-PCR hybridization
with the internal probe specific to S. aureus (SEQ ID NO. 587)
described in Example 7 and the internal probes specific to
wild-type S. aureus gyrA and parC (SEQ ID NOs. 1940-1941) and to
the S. aureus gyrA and parC variants (SEQ ID NOs. 1333-1338).
[0388] An assay was also developed for the detection of
quinolone-resistant S. aureus using the SmartCycler (Cepheid).
Real-time detection is based on the use of S. aureus parC-specific
primers (SEQ ID NOs. 1342 and 1343) and the Staphylococcus-specific
primers (SEQ ID NOs. 553 and 575) described in Example 7. Internal
probes were designed for molecular beacon detection of the
wild-type S. aureus parC (SEQ ID NO. 1939), for detection of the
Ser-80 to Tyr or Phe amino acid substitutions in the ParC subunit
encoded by S. aureus parC (SEQ ID NOs. 1938 and 1955) and for
detection of S. aureus (SEQ ID NO. 2282).
Example 34
Development of a PCR Assay for the Detection and Identification of
Klebsiella pneumoniae and its Quinolone Resistance Genes gyrA and
parC
[0389] The analysis of gyrA and parC sequences from a variety of
bacterial species from the public databases and from the database
described in Example 32 revealed conserved regions allowing the
design of PCR primers specific to the
quinolone-resistance-determining region (QRDR) of gyrA and parC
from K. pneumoniae. PCR primer pair SEQ ID NOs. 1936 and 1937, or
pair SEQ ID NOs. 1937 and 1942, were designed to amplify the gyrA
sequence of K. pneumoniae, whereas PCR primer pair SEQ ID NOs. 1934
and 1935 was designed to amplify K. pneumoniae parC sequence. An
alternative pair, SEQ ID NOs. 1935 and 1936, can also amplify K.
pneumoniae parC. The comparison of gyrA and parC sequences from K.
pneumoniae strains with various levels of quinolone resistance
allowed the identification of amino acid substitutions Ser-83 to
Tyr or Phe and Asp-87 to Gly or Ala and Asp-87 to Asn in the GyrA
subunit of DNA gyrase encoded by gyrA and amino acid changes Ser-80
to Ile or Arg and Glu-84 to Gly or Lys in the ParC subunit of
topoisomerase IV encoded by parC. These amino acid substitutions in
the GyrA and ParC subunits occur in isolates with intermediate- or
high-level quinolone resistance. Internal probes for the specific
detection of wild-type K. pneumoniae gyrA (SEQ ID NO. 1943) and
wild-type K. pneumoniae parC (SEQ ID NO. 1944) as well as internal
probes for the specific detection of each of the gyrA (SEQ ID NOs.
1945-1949) and parC mutations identified in quinolone-resistant K.
pneumoniae (SEQ ID NOs. 1950-1953) were designed.
[0390] Two multiplex using the K. pneumoniae gyrA- and
parC-specific primer pairs were used: the first multiplex contained
K. pneumoniae gyrA-specific primers (SEQ ID NOs. 1937 and 1942) and
K. pneumoniae parC-specific primers (SEQ ID NOs. 1934 and 1935) and
the second multiplex contained K. pneumoniae gyrA/parC-specific
primer (SEQ ID NOs. 1936), K. pneumoniae gyrA-specific primer (SEQ
ID NO. 1937) and K. pneumoniae parC-specific primer (SEQ ID NO.
1935). Standard PCR was carried out on PTC-200 thermocyclers (MJ
Research) using for the first multiplex 0.6, 0.6, 0.4, 0.4 .mu.M of
each primer, respectively, and for the second multiplex 0.8, 0.4,
0.4 .mu.M of each primer, respectively. PCR amplification and
agarose gel electrophoresis of the amplified products were
performed as described in Example 28. The optimal cycling
conditions for maximum sensitivity and specificity were 3 minutes
at 95.degree. C. for initial denaturation, then forty cycles of two
steps consisting of 1 second at 95.degree. C. and 30 seconds at
62.degree. C., followed by terminal extension at 72.degree. C. for
2 minutes. The specificity of the two multiplex assays with
40-cycle PCR was verified by using 0.1 ng of purified genomic DNA
from a panel of gram-negative bacteria. The list included:
Acinetobacter baumannii, Citrobacter freundii, Eikenella corrodens,
Enterobacter aerogenes, Enterobacter cancerogenes, Enterobacter
cloacae, Escherichia coli (10 strains), Haemophilus influenzae,
Klebsiella pneumoniae, Klebsiella ornitholytica, Klebsiella oxytoca
(2 strains), Klebsiella planticola, Klebsiella terrigena, Kluyvera
ascorbata, Kluyvera cryocrescens, Kluyvera georgiana, Neisseria
gonorrhoeae, Proteus mirabilis, Proteus vulgaris, Pseudomonas
aeruginosa, Salmonella choleraesuis subsp. typhimurium, Salmonella
enteritidis, Serratia liquefaciens, Serratia marcescens and
Yersinia enterocolytica. For both multiplex, strong amplification
of both gyrA and parC was observed only for the K. pneumoniae
strain tested. The sensitivity of the two multiplex assays with
40-cycle PCR was verified with one quinolone-sensitive strain of K.
pneumoniae. The detection limit was around 10 copies of genomic
DNA.
[0391] The complete assay for the specific detection of K.
pneumoniae and its susceptibility to quinolone contains the
Klebsiella-specific primers (SEQ ID NOs. 1331 and 1332) described
in Example 29 and either the multiplex containing the K. pneumoniae
gyrA- and parC-specific primers (SEQ ID NOs. 1935, 1936, 1937) or
the multiplex containing the K. pneumoniae gyrA- and parC-specific
primers (SEQ ID NOs. 1934, 1937, 1939, 1942). Amplification is
coupled with post-PCR hybridization with the internal probe
specific to K. pneumoniae (SEQ ID NO. 2167) described in Example 29
and the internal probes specific to wild-type K. pneumoniae gyrA
and parC (SEQ ID NOs. 1943, 1944) and to the K. pneumoniae gyrA and
parC variants (SEQ ID NOs. 1945-1949 and 1950-1953).
[0392] An assay was also developed for the detection of
quinolone-resistant K. pneumoniae using the SmartCycler (Cepheid).
Real-time detection is based on the use of resistant K. pneumoniae
gyrA-specific primers (SEQ ID NOs. 1936 and 1937) and the K.
pneumoniae-specific primers (SEQ ID NOs. 1331 and 1332) described
in Example 29. Internal probes were designed for molecular beacon
detection of the wild-type K. pneumoniae gyrA (SEQ ID NO. 2251),
for detection of the Ser-83 to Tyr or Phe and/or Asp-87 to Gly or
Asn in the GyrA subunit of DNA gyrase encoded by gyrA (SEQ ID NOs.
2250) and for detection of K. pneumoniae (SEQ ID NO. 2281).
Example 35
Development of a PCR Assay for Detection and Identification of S.
pneumoniae and its Quinolone Resistance Genes gyrA and parC
[0393] The analysis of gyrA and parC sequences from a variety of
bacterial species revealed conserved regions allowing the design of
PCR primers able to amplify the quinolone-resistance-determining
region (QRDR) of gyrA and parC from all S. pneumoniae strains. PCR
primer pair SEQ ID NOs. 2040 and 2041 was designed to amplify the
QRDR of S. pneumoniae gyrA, whereas PCR primer pair SEQ ID NOs.
2044 and 2045 was designed to amplify the QRDR of S. pneumoniae
parC. The comparison of gyrA and parC sequences from S. pneumoniae
strains with various levels of quinolone resistance allowed the
identification of amino acid substitutions Ser-81 to Phe or Tyr in
the GyrA subunit of DNA gyrase encoded by gyrA and amino acid
changes Ser-79 to Phe in the ParC subunit of topoisomerase IV
encoded by parC. These amino acid substitutions in the GyrA and
ParC subunits occur in isolates with intermediate- or high-level
quinolone resistance. Internal probes for the specific detection of
each of the gyrA (SEQ ID NOs. 2042 and 2043) and parC (SEQ ID NO.
2046) mutations identified in quinolone-resistant S. pneumoniae
were designed.
[0394] For all bacterial species, amplification was performed from
purified genomic DNA. 1 .mu.l of genomic DNA at 0.1 ng/.mu.L was
transferred directly to a 19 .mu.l PCR mixture. Each PCR reaction
contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100,
2.5 mM MgCl.sub.2, 0.4 .mu.M (each) of the above primers SEQ ID
NOs. 2040, 2041, 2044 and 2045, 0.05 mM bovine serum albumin (BSA)
and 0.5 U Taq polymerase coupled with TaqStart.TM. antibody. The
optimal cycling conditions for maximum sensitivity' and specificity
were 3 minutes at 95.degree. C. for initial denaturation, then
forty cycles of two steps consisting of 1 second at 95.degree. C.
and 30 seconds at 58.degree. C., followed by terminal extension at
72.degree. C. for 2 minutes. In order to generate Digoxigenin
(DIG)-labeled amplicons for capture probe hybridization,
0.1.times.PCR DIG labeling four deoxynucleoside triphosphates mix
(Boehringer Mannheim GmbH) was used for amplification.
[0395] The DIG-labeled amplicons were hybridized to the capture
probes bound to 96-well plates. The plates were incubated with
anti-DIG-alkaline phosphatase and the chemiluminescence was
measured by using a luminometer (MLX, Dynex Technologies Inc.)
after incubation with CSPD and recorded as Relative Light Unit
(RLU). The RLU ratio of tested sample with and without captures
probes was then calculated. A ratio 2.0 was defined as a positive
hybridization signal. All reactions were performed in
duplicate.
[0396] The specificity of the multiplex assay with 40-cycle PCR was
verified by using 0.1 ng of purified genomic DNA from a panel of
bacteria listed in Table 13. Strong amplification of both gyrA and
parC was detected only for the S. pneumoniae strains tested. Weak
amplification of both gyrA and parC genes was detected for
Staphylococcus simulans. The detection limit tested with purified
genomic DNA from 5 strains of S. pneumoniae was 1 to 10 genome
copies. In addition, 5 quinolone-resistant and 2
quinolone-sensitive clinical isolates of S. pneumoniae were tested
to further validate the developed multiplex PCR coupled with
capture probe hybridization assays. There was a perfect correlation
between detection of S. pneumoniae gyrA and parC mutations and the
susceptibility to quinolone.
[0397] The complete assay for the specific detection of S.
pneumoniae and its susceptibility to quinolone contains the S.
pneumoniae-specific primers (SEQ ID NOs. 1179 and 1181) described
in Example 20 and the multiplex containing the S. pneumoniae
gyrA-specific and parC-specific primer pairs (SEQ ID NOS. 2040 and
2041 and SEQ ID NOs. 2044 and 2045). Amplification is coupled with
post-PCR hybridization with the internal probe specific to S.
pneumoniae (SEQ ID NO. 1180) described in Example and the internal
probes specific to each of the S. pneumoniae gyrA and parC variants
(SEQ ID NOs. 2042, 2043 and 2046).
Example 36
Detection of Extended-Spectrum TEM-Type .beta.-Lactamases in
Escherichia coli
[0398] The analysis of TEM sequences which confer resistance to
third-generation cephalosporins and to .beta.-lactamase inhibitors
allowed the identification of amino acid substitutions Met-69 to
Ile or Leu or Val, Ser-130 to Gly, Arg-164 to Ser or His, Gly-238
to Ser, Glu-240 to Lys and Arg-244 to Ser or Cys or Thr or His or
Leu. PCR primers SEQ ID NOs. 1907 and 1908 were designed to amplify
TEM sequences. Internal probes for the specific detection of
wild-type TEM (SEQ ID NO. 2141) and for each of the amino acid
substitutions (SEQ ID NOs. 1909-1926) identified in TEM variants
were designed to detect resistance to third-generation
cephalosporins and to .beta.-lactamase inhibitors. Design and
synthesis of primers and probes, and detection of the hybridization
were performed as described in Example 7.
[0399] For all bacterial species, amplification was performed from
purified genomic DNA. One .mu.l of genomic DNA at 0.1 ng/.mu.l was
transferred directly to a 19 .mu.l PCR mixture. Each PCR reaction
contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0); 0.1% Triton X-100,
2.5 mM MgCl.sub.2, 0.4 .mu.M of the TEM-specific primers SEQ ID
NOs. 1907 and 1908, 200 .mu.M (each) of the four deoxynucleoside
triphosphates, 0.05 mM bovine serum albumin (BSA) and 0.5 U Taq
polymerase (Promega) coupled with TaqStart.TM. antibody. PCR
amplification and agarose gel analysis of the amplified products
were performed as described in Example 28. The optimal cycling
conditions for maximum sensitivity and specificity were 3 minutes
at 95.degree. C. for initial denaturation, then forty cycles of
three steps consisting of 5 seconds at 95.degree. C., 30 seconds at
55.degree. C. and 30 seconds at 72.degree. C., followed by terminal
extension at 72.degree. C. for 2 minutes.
[0400] The specificity of the TEM-specific primers with 40-cycle
PCR was verified by using 0.1 ng of purified genomic from the
following bacteria: three third-generation cephalosporin-resistant
Escherichia coli strains (one with TEM-10, one with TEM-28 and the
other with TEM-49), two third-generation cephalosporin-sensitive
Escherichia coli strain (one with TEM-1 and the other without TEM),
one third-generation cephalosporin-resistant Klebsiella pneumoniae
strain (with TEM-47), and one .beta.-lactamase-inhibitor-resistant
Proteus mirabilis strain (with TEM-39). Amplification with the
TEM-specific primers was detected only for strains containing
TEM.
[0401] The sensitivity of the assay with 40-cycle PCR was verified
with three E. coli strains containing TEM-1 or TEM-10 or TEM-49,
one K. pneumoniae strain containing TEM-47 and one P. mirabilis
strain containing TEM-39. The detection limit was 5 to 100 copies
of genomic DNA, depending on the TEM-containing strains tested.
[0402] The TEM-specific primers SEQ ID NOs. 1907 and 1908 were used
in multiplex with the Escherichia coli/Shigella sp.-specific
primers SEQ ID NOs. 1661 and 1665 described in Example 28 to allow
the complete identification of Escherichia coli/Shigella sp. and
the susceptibility to .beta.-lactams. PCR amplification with 0.4
.mu.M of each of the primers and agarose gel analysis of the
amplified products was performed as described above.
[0403] The specificity of the multiplex with 40-cycle PCR was
verified by using 0.1 ng of purified genomic DNA from the following
bacteria: three third-generation cephalosporin-resistant
Escherichia coli strains (one with TEM-10, one with TEM-28 and the
other with TEM-49), two third-generation cephalosporin-sensitive
Escherichia coli strain (one with TEM-1 and the other without TEM),
one third-generation cephalosporin-resistant Klebsiella pneumoniae
strain (with TEM-47), and one .beta.-lactamase-inhibitor-resistant
Proteus mirabilis strain (with TEM-39). The multiplex was highly
specific to Escherichia coli strains containing TEM.
[0404] The complete assay for detection of TEM-type
.beta.-lactamases in E. coli includes PCR amplification using the
multiplex containing the TEM-specific primers (SEQ ID NOs. 1907 and
1908) and the Escherichia coli/Shigella sp.-specific primers (SEQ
ID NOs. 1661 and 1665) coupled with post PCR-hybridization with the
internal probes specific to wild-type TEM (SEQ ID NO. 2141) and to
the TEM variants (SEQ ID NOs. 1909-1926).
Example 37
Detection of Extended-Spectrum SHY-Type .beta.-Lactamases in
Klebsiella pneumoniae
[0405] The comparison of SHV sequences, which confer resistance to
third-generation cephalosporins and to .beta.-lactamase inhibitors,
allowed the identification of amino acid substitutions Ser-130 to
Gly, Asp-179 to Ala or Asn, Gly-238 to Ser, and Glu-240 to Lys. PCR
primer pair SEQ ID NOs. 1884 and 1885 was designed to amplify SHV
sequences. Internal probes for the specific identification of
wild-type SHV (SEQ ID NO. 1896) and for each of the amino acid
substitutions (SEQ ID NOs. 1886-1895 and 1897-1898) identified in
SHV variants were designed to detect resistance to third-generation
cephalosporins and to .beta.-lactamase inhibitors. Design and
synthesis of primers and probes, and detection of the hybridization
were performed as described in Example 7.
[0406] For all bacterial species, amplification was performed from
purified genomic DNA. One .mu.l of genomic DNA at 0.1 ng/.mu.l was
transferred directly to a 19 .mu.l PCR mixture. Each PCR reaction
contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100,
2.5 mM MgCl.sub.2, 0.4 .mu.M of the SHV-specific primers SEQ ID NO.
1884 and 1885, 200 .mu.M (each) of the four deoxynucleoside
triphosphates, 0.05 mM bovine serum albumin (BSA) and 0.5 U Taq
polymerase (Promega) coupled with TaqStart.TM. antibody. PCR
amplification and agarose gel analysis of the amplified products
were performed as described in Example 28. The optimal cycling
conditions for maximum sensitivity and specificity were 3 minutes
at 95.degree. C. for initial denaturation, then forty cycles of
three steps consisting of 5 seconds at 95.degree. C., 30 seconds at
55.degree. C. and 30 seconds at 72.degree. C., followed by terminal
extension at 72.degree. C. for 2 minutes.
[0407] The specificity of the SHV-specific primers with 40-cycle
PCR was verified by using 0.1 ng of purified genomic from the
following bacteria: two third-generation cephalosporin-resistant
Klebsiella pneumoniae strains (one with SHV-2a and the other with
SHV-12), one third-generation cephalosporin-sensitive Klebsiella
pneumoniae strain (with SHV-1), two third-generation
cephalosporin-resistant Escherichia coli strains (one with SHV-8
and the other with SHV-7), and two third-generation
cephalosporin-sensitive Escherichia coli strains (one with SHV-1
and the other without any SHV). Amplification with the SHV-specific
primers was detected only for strains containing SHV.
[0408] The sensitivity of the assay with 40-cycle PCR was verified
with four strains containing SHV. The detection limit was 10 to 100
copies of genomic DNA, depending on the SHV-containing strains
tested.
[0409] The amplification was coupled with post-PCR hybridization
with the internal probes specific for identification of wild-type
SHV (SEQ ID NO. 1896) and for each of the amino acid substitutions
(SEQ ID NOs. 1886-1895 and 1897-1898) identified in SHV variants.
The specificity of the probes was verified with six strains
containing various SHV enzymes, one Klebsiella pneumoniae strain
containing SHV-1, one Klebsiella pneumoniae strain containing
SHV-2a, one Klebsiella pneumoniae strain containing SHV-12, one
Escherichia coli strain containing SHV-1, one Escherichia coli
strain containing SHV-7 and one Escherichia coli strain containing
SHV-8. The probes correctly detected each of the SHV genes and
their specific mutations. There was a perfect correlation between
the SHV genotype of the strains and the susceptibility to
.beta.-lactam antibiotics.
[0410] The SHV-specific primers SEQ ID NOs. 1884 and 1885 were used
in multiplex with the K. pneumoniae-specific primers SEQ ID NOs.
1331 and 1332 described in Example 29 to allow the complete
identification of K. pneumoniae and the susceptibility to
.beta.-lactams. PCR amplification with 0.4 .mu.M of each of the
primers and agarose gel analysis of the amplified products were
performed as described above.
[0411] The specificity of the multiplex with 40-cycle PCR was
verified by using 0.1 ng of purified genomic DNA from the following
bacteria: three K. pneumoniae strains containing SHV-1, one
Klebsiella pneumoniae strain containing SHV-2a, one Klebsiella
pneumoniae strain containing SHV-12, one K. rhinoscleromatis strain
containing SHV-1, one Escherichia coli strain without SHV. The
multiplex was highly specific to Klebsiella pneumoniae strain
containing SHV.
Example 38
Development of a PCR Assay for the Detection and Identification of
Neisseria gonorrhoeae and its associated tetracycline resistance
gene tetM
[0412] The analysis of publicly available tetM sequences revealed
conserved regions allowing the design of PCR primers specific to
tetM sequences. The PCR primer pair SEQ ID NOs. 1588 and 1589 was
used in multiplex with the Neisseria gonorrhoeae-specific primers
SEQ ID NOs. 551 and 552 described in Example 31. Sequence alignment
analysis of tetM sequences revealed regions suitable for the design
of an internal probe specific to tetM (SEQ ID NO. 2254). PCR
amplification was carried out on PTC-200 thermocyclers (MJ
Research) using 0.4 .mu.M of each primer pair as described in
Example 28. The optimal cycling conditions for maximum sensitivity
and specificity were as follow: three minutes at 95.degree. C. for
initial denaturation, then forty cycles of two steps consisting of
1 second at 95.degree. C. and 30 seconds at 60.degree. C., followed
by terminal extension at 72.degree. C. for 2 minutes.
[0413] The specificity of the multiplex PCR assay with 40-cycle PCR
was verified by using 0.1 ng of purified genomic DNA from the
following bacteria: two tetracycline-resistant Escherichia coli
strains (one containing the tetracycline-resistant gene tetB and
the other containing the tetracycline-resistant gene tetC), one
tetracycline-resistant Pseudomonas aeruginosa strain (containing
the tetracycline-resistant gene tetA), nine tetracycline-resistant
Neisseria gonorrhoeae strains, two tetracycline-sensitive Neisseria
meningitidis strains, one tetracycline-sensitive Neisseria
polysaccharea strain, one tetracycline-sensitive Neisseria sicca
strain and one tetracycline-sensitive Neisseria subflava strain.
Amplification with both the tetM-specific and Neisseria
gonorrhoeae-specific primers was detected only for N. gonorrhoeae
strains containing tetM. There was a weak amplification signal
using Neisseria gonorrhoeae-specific primers for the following
species: Neisseria sicca, Neisseria polysaccharea and Neisseria
meningitidis. There was a perfect correlation between the tetM
genotype and the tetracycline susceptibility pattern of the
Neisseria gonorrhoeae strains tested. The internal probe specific
to N. gonorrhoeae SEQ ID NO. 2166 described in Example 31 can
discriminate Neisseria gonorrhoeae from the other Neisseria sp.
[0414] The sensitivity of the assay with 40-cycle PCR was verified
with two tetracycline resistant strains of N. gonorrhoeae. The
detection limit was 5 copies of genomic DNA for both strains.
Example 39
Development of a PCR Assay for the Detection and Identification of
Shigella Sp. and their Associated Trimethoprim Resistance Gene
dhfrIa
[0415] The analysis of publicly available dhfrIa and other dhfr
sequences revealed regions allowing the design of PCR primers
specific to dhfrIa sequences. The PCR primer pair (SEQ ID NOs. 1459
and 1460) was used in multiplex with the Escherichia coli/Shigella
sp.-specific primers SEQ ID NOs. 1661 and 1665 described in Example
28. Sequence alignment analysis of dhfrIa sequences revealed
regions suitable for the design of an internal probe specific to
dhfrIa (SEQ ID NO. 2253). PCR amplification and agarose gel
analysis of the amplified products were performed as described in
Example 28 with an annealing temperature of 60.degree. C. The
specificity of the multiplex assay with 40-cycle PCR was verified
by using 0.1 ng of purified genomic DNA from a panel of bacteria.
The list included the following trimethoprim-sensitive strains,
Salmonella typhimyurium, Salmonella typhi, Salmonella enteritidis,
Tatumella ptyseos, Klebsiella pneumoniae, Enterobacter aerogenes,
Citrobacter farmeri, Campylobacter jejuni, Serratia marcescens,
Shigella dysenteriae, Shigella flexneri, Shigella sonnei, six
trimethoprim-resistant Escherichia coli strains (containing dhfrIa
or dhfrV or dhfrVII or dhfrXII or dhfrXIII or dhfrXV), four
trimethoprim-resistant strains containing dhfrIa (Shigella sonnei,
Shigella flexneri, Shigella dysenteriae and Escherichia coli).
There was a perfect correlation between the dhfrIa genotype and the
trimethoprim susceptibility pattern of the Escherichia coli and
Shigella sp. strains tested. The dhfrIa primers were specific to
the dhfrIa gene and did not amplify any of the other
trimethoprim-resistant dhfr genes tested. The sensitivity of the
multiplex assay with 40-cycle PCR was verified with three strains
of trimethoprim-resistant strains of Shigella sp. The detection
limit was 5 to 10 genome copies of DNA, depending on the Shigella
sp. strains tested.
Example 40
Development of a PCR Assay for the Detection and Identification of
Acinetobacter baumannii and its Associated Aminoglycoside
Resistance Gene aph(3')-VIa
[0416] The comparison of publicly available aph(3')-VIa sequence
revealed regions allowing the design of PCR primers specific to
aph(3)-VIa. The PCR primer pair (SEQ ID NOs. 1404 and 1405) was
used in multiplex with the Acinetobacter baumannii-specific primers
SEQ ID NOs. 1692 and 1693 described in Example 30. Analysis of the
aph(3)-VIa sequence revealed region suitable for the design of an
internal probe specific to aph(3')-VIa (SEQ ID NO. 2252). PCR
amplification and agarose gel analysis of the amplified products
were performed as described in Example 28. The specificity of the
multiplex assay with 40-cycle PCR was verified by using 0.1 ng of
purified genomic DNA from a panel of bacteria including: two
aminoglycoside-resistant A. baumanni strains (containing
aph(3)-VIa), one aminoglycoside-sensitive A. baumani strain, one of
each of the following aminoglycoside-resistant bacteria, one
Serratia marcescens strain containing the aminoglycoside-resistant
gene aacC1, one Serratia marcescens strain containing the
aminoglycoside-resistant gene aacC4, one Enterobacter cloacae
strain containing the aminoglycoside-resistant gene aacC2, one
Enterococcus faecalis containing the aminoglycoside-resistant gene
aacA-aphD, one Pseudomonas aeruginosa strain containing the
aminoglycoside-resistant gene aac6IIa and one of each of the
following aminoglycoside-sensitive bacterial species, Acinetobacter
anitratus, Acinetobacter lwoffi, Psychobbacter phenylpyruvian,
Neisseria gonorrhoeae, Haemophilus haemolyticus, Haemophilus
influenzae, Yersinia enterolitica, Proteus vulgaris, Eikenella
corrodens, Escherichia coli. There was a perfect correlation
between the aph(3')-VIa genotype and the aminoglycoside
susceptibility pattern of the A. baumannii strains tested. The
aph(3)-VIa-specific primers were specific to the aph(3)-VIa gene
and did not amplify any of the other aminoglycoside-resistant genes
tested. The sensitivity of the multiplex assay with 40-cycle PCR
was verified with two strains of aminoglycoside-resistant strains
of A. baumannii. The detection limit was 5 genome copies of DNA for
both A. baumannii strains tested.
Example 41
Specific Identification of Bacteroides fragilis Using atpD (V-Type)
Sequences
[0417] The comparison of atpD (V-type) sequences from a variety of
bacterial species allowed the selection of PCR primers for
Bacteroides fragilis. The strategy used to design the PCR primers
was based on the analysis of a multiple sequence alignment of
various atpD sequences from B. fragilis, as well as atpD sequences
from the related species B. dispar, bacterial genera and archaea,
especially representatives with phylogenetically related atpD
sequences. A careful analysis of this alignment allowed the
selection of oligonucleotide sequences which are conserved within
the target species but which discriminate sequences from other
species, especially from closely related species B. dispar, thereby
permitting the species-specific and ubiquitous detection and
identification of the target bacterial species.
[0418] The chosen primer pair, SEQ ID NOs. 2134-2135, produces an
amplification product of 231 bp. Standard PCR was carried out on
PTC-200 thermocyclers (MJ Research Inc.) using 0.4 .mu.M of each
primers pair as described in Example 28. The optimal cycling
conditions for maximum sensitivity and specificity were as follows:
three minutes at 95.degree. C. for initial denaturation, then forty
cycles of two steps consisting of 1 second at 95.degree. C. and 30
seconds at 60.degree. C., followed by terminal extension at
72.degree. C. for 2 minutes.
[0419] The format of this assay is not limited to the one described
above. A person skilled in the art could adapt the assay for
different formats such as PCR with real-time detection using
molecular beacon probes. Molecular beacon probes designed to be
used in this assay include, but are not limited to, SEQ ID NO. 2136
for the detection of the B. fragilis amplicon.
Example 42
Evidence for Horizontal Gene Transfer in the Evolution of the
Elongation Factor Tu in Enterococci
Overview
[0420] The elongation factor Tu, encoded by tuf genes, is a GTP
binding protein that plays a central role in protein synthesis. One
to three tuf genes per genome are present depending on the
bacterial species. Most low G+C gram-positive bacteria carry only
one tuf gene. We have designed degenerate PCR primers derived from
consensus sequences of the tuf gene to amplify partial tuf
sequences from 17 enterococcal species and other phylogenetically
related species. The amplified DNA fragments were sequenced either
by direct sequencing or by sequencing cloned inserts containing
putative amplicons. Two different tuf genes (tufA and tufB) were
found in 11 enterococcal species, including Enterococcus avium, E.
casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E.
hirae, E. malodoratus, E. mundtii, E. pseudoavium, and E.
raffinosus. For the other six enterococcal species (E. cecorum, E.
columbae, E. faecalis, E. sulfureus, E. saccharolyticus, and E.
solitarius), only the tufA gene was present. Based on 16S rRNA gene
sequence analysis, the 11 species having two tuf genes all share a
common ancestor, while the six species having only one copy
diverged from the enterococcal lineage before that common ancestor.
The presence of one or two copies of the tuf gene in enterococci
was confirmed by Southern hybridization. Phylogenetic analysis of
tuf sequences demonstrated that the enterococcal tufA gene branches
with the Bacillus, Listeria and Staphylococcus genera, while the
enterococcal tufB gene clusters with the genera Streptococcus and
Lactococcus. Primary structure, analysis showed that four amino
acid residues within the sequenced regions are conserved and unique
to the enterococcal tufB genes and the tuf genes of streptococci
and L. lactis. The data suggest that an ancestral streptococcus or
a streptococcus-related species may have horizontally transferred a
tuf gene to the common ancestor of the 11 enterococcal species
which now carry two tuf genes.
Introduction
[0421] The elongation factor Tu (EF-Tu) is a GTP binding protein
playing a central role in protein synthesis. It mediates the
recognition and transport of aminoacyl-tRNAs and their positioning
to the A-site of the ribosome. The highly conserved function and
ubiquitous distribution render the elongation factor a valuable
phylogenetic marker among eubacteria and even throughout the
archaebacterial and eukaryotic kingdoms. The tuf genes encoding
elongation factor Tu are present in various copy numbers per
bacterial genome. Most gram-negative bacteria contain two tuf
genes. As found in Escherichia coli, the two genes, while being
almost identical in sequence, are located in different parts of the
bacterial chromosome. However, recently completed microbial genomes
revealed that only one tuf gene is found in Helicobacter pylori as
well as in some obligate parasitic bacteria, such as Borrelia
burgdorferi, Rickettsia prowazekii, and Treponema pallidum, and in
some cyanobacteria. In most gram-positive bacteria studied so far,
only one tuf gene was found. However, Southern hybridization showed
that there are two tuf genes in some clostridia as well as in
Streptomyces coelicolor and S. lividans. Up to three tuf-like genes
have been identified in S. ramocissimus.
[0422] Although massive prokaryotic gene transfer is suggested to
be one of the factors responsible for the evolution of bacterial
genomes, the genes encoding components of the translation machinery
are thought to be highly conserved and difficult to be transferred
horizontally due to the complexity of their interactions. However,
a few recent studies demonstrated evidence that horizontal gene
transfer has also occurred in the evolution of some genes coding
for the translation apparatus, namely, 16S rRNA and some
aminoacyl-tRNA synthetases. No further data suggest that such a
mechanism is involved in the evolution of the elongation factors.
Previous studies concluded that the two copies of tuf genes in the
genomes of some bacteria resulted from an ancient event of gene
duplication. Moreover, a study of the tuf gene in R. prowazekii
suggested that intrachromosomal recombination has taken place in
the evolution of the genome of this organism.
[0423] To date, little is known about the tuf genes of enterococcal
species. In this study, we analyzed partial sequences of tuf genes
in 17 enterococcal species, namely, E. avium, E. casseliflavus, E.
cecorum, E. columbae, E. dispar, E. durans, E. faecalis, E.
faecium, E. gallinarum, E. hirae, E. malodoratus, E. mundtii, E.
pseudoavium, E. raffinosus, E. saccharolyticus, E. solitarius, and
E. sulfureus. We report here the presence of two divergent copies
of tuf genes in 11 of these enterococcal species. The 6 other
species carried a single tuf gene. The evolutionary implications
are discussed.
Materials and Methods
[0424] Bacterial strains. Seventeen enterococcal strains and other
gram-positive bacterial strains obtained from the American Type
Culture Collection (ATCC, Manassas, Va.) were used in this study
(Table 16). All strains were grown on sheep blood agar or in
brain-heart infusion broth prior to DNA isolation.
[0425] DNA isolation. Bacterial DNAs were prepared using the G NOME
DNA extraction kit (Bio101, Vista, Calif.) as previously
described.
[0426] Sequencing of putative tuf genes. In order to obtain the tuf
gene sequences of enterococci and other gram-positive bacteria, two
sequencing approaches were used: 1) sequencing of cloned PCR
products and 2) direct sequencing of PCR products. A pair of
degenerate primers (SEQ ID NOs. 664 and 697) were used to amplify
an 886-bp portion of the tuf genes from enterococcal species and
other gram-positive bacteria as previously described. For E. avium,
E. casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum,
E. hirae, E. mundtii, E. pseudoavium, and E. raffinosus, the
amplicons were cloned using the Original TA cloning kit
(Invitrogen, Carlsbad, Calif.) as previously described. Five clones
for each species were selected for sequencing. For E. cecorum, E.
faecalis, E. saccharolyticus, and E. solitarius as well as the
other gram-positive bacteria, the sequences of the 886-bp amplicons
were obtained by direct sequencing. Based on the results obtained
from the earlier rounds of sequencing, two pairs of primers were
designed for obtaining the partial tuf sequences from the other
enterococcal species by direct sequencing. One pair of primers (SEQ
ID NOs. 543 and 660) were used to amplify the enterococcal tuf gene
fragments from E. columbae, E. malodoratus, and E. sulfureus.
Another pair of primers (SEQ ID NOs. 664 and 661) were used to
amplify the second tuf gene fragments from E. avium, E.
malodoratus, and E. pseudoavium.
[0427] Prior to direct sequencing, PCR products were
electrophoresed on 1% agarose gel at 120V for 2 hours. The gel was
then stained with 0.02% methylene blue for 30 minutes and washed
twice with autoclaved distilled water for 15 minutes. The gel
slices containing PCR products of the expected sizes were cut out
and purified with the QIAquick gel extraction kit (QIAgen Inc.,
Mississauga, Ontario, Canada) according to the manufacturer's
instructions. PCR mixtures for sequencing were prepared as
described previously. DNA sequencing was carried out with the Big
Dye.TM. Terminator Ready Reaction cycle sequencing kit using a 377
DNA sequencer (PE Applied Biosystems, Foster City, Calif.). Both
strands of the amplified DNA were sequenced. The sequence data were
verified using the Sequencer.TM. 3.0 software (Gene Codes Corp.,
Ann Arbor, Mich.).
[0428] Sequence analysis and phylogenetic study. Nucleotide
sequences of the tuf genes and their respective flanking regions
for E. faecalis, Staphylococcus aureus, and Streptococcus
pneumoniae, were retrieved from the TIGR microbial genome database
and S. pyogenes from the University of Oklahoma database. DNA
sequences and deduced protein sequences obtained in this study were
compared with those in all publicly available databases using the
BLAST and FASTA programs. Unless specified, sequence analysis was
conducted with the programs from GCG package (Version 10; Genetics
Computer Group, Madison, Wis.). Sequence alignment of the tuf genes
from 74 species representing all three kingdoms of life (Tables 16
and 17) were carried out by use of Pileup and corrected upon visual
analysis. The N- and C-termini extremities of the sequences were
trimmed to yield a common block of 201 amino acids sequences and
equivocal residues were removed. Phylogenetic analysis was
performed with the aid of PAUP 4.0b4 written by Dr. David L.
Swofford (Sinauer Associates, Inc., Publishers, Sunderland, Mass.).
The distance matrix and maximum parsimony were used to generate
phylogenetic trees and bootstrap resampling procedures were
performed using 500 and 100 replications in each analysis,
respectively.
[0429] Protein structure analysis. The crystal structures of
(i)Thermus aquaticus EF-Tu in complex with Phe-tRNA.sup.Phe and a
GTP analog and (ii) E. coli EF-Tu in complex with GDP served as
templates for constructing the equivalent models for enterococcal
EF-Tu. Homology modeling of protein structure was performed using
the SWISS-MODEL server and inspected using the SWISS-PDB viewer
version 3.1.
[0430] Southern hybridization. In a previous study, we amplified
and cloned an 803-bp PCR product of the tuf gene fragment from E.
faecium. Two divergent sequences of the inserts, which we assumed
to be tufA and tufB genes, were obtained. The recombinant plasmid
carrying either tufA or tufB sequence was used to generate two
probes labeled with Digoxigenin (DIG)-11-dUTP by PCR incorporation
following the instructions of the manufacturer (Boehringer
Mannheim, Laval, Quebec, Canada). Enterococcal genomic DNA samples
(1-2 .mu.g) were digested to completion with restriction
endonucleases BglII and XbaI as recommended by the supplier
(Amersham Pharmacia Biotech, Mississauga, Ontario, Canada). These
restriction enzymes were chosen because no restriction sites were
observed within the amplified tuf gene fragments of most
enterococci. Southern blotting and filter hybridization were
performed using positively charged nylon membranes (Boehringer
Mannheim) and QuikHyb hybridization solution (Stratagene Cloning
Systems, La Jolla, Calif.) according to the manufacturers'
instructions with modifications. Twenty .mu.l of each digestion
were electrophoresed for 2 h at 120V on a 0.8% agarose gel. The DNA
fragments were denatured with 0.5 M NaOH and transferred by
Southern blotting onto a positively charged nylon membrane
(Boehringer Mannheim). The filters were pre-hybridized for 15 min
and then hybridized for 2 h in the QuikHyb solution at 68.degree.
C. with either DIG-labeled probe. Posthybridization washings were
performed twice with 0.5.times.SSC, 1% SDS at room temperature for
15 min and twice in the same solution at 60.degree. C. for 15 min.
Detection of bound probes was achieved using disodium
3-(4-methoxyspiro(1,2-dioxetane-3,2'-(5'-chloro)
tricyclo(3,3.1.1.sup.3.7) decan)-4-yl) phenyl phosphate (CSPD)
(Boehringer Mannheim) as specified by the manufacturer.
[0431] GenBank submission. The GenBank accession numbers for
partial tuf gene sequences generated in this study are given in
Table 16.
Results
[0432] Sequencing and nucleotide sequence analysis. In this study,
all gram-positive bacteria other than enterococci yielded a single
tuf sequence of 886 by using primers SEQ ID NOs. 664 and 697 (Table
16). Each of four enterococcal species including E. cecorum, E.
faecalis, E. saccharolyticus, and E. solitarius also yielded one
886-bp tuf sequence. On the other hand, for E. avium, E.
casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E.
hirae, E. mundtii, E. pseudoavium, and E. raffinosus, direct
sequencing of the 886-bp fragments revealed overlapping peaks
according to their sequence chromatograms, suggesting the presence
of additional copies of the tuf gene. Therefore, the tuf gene
fragments of these 10 species were cloned first and then sequenced.
Sequencing data revealed that two different types of tuf sequences
(tufA and tufB) are found in eight of these species including E.
casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E.
hirae, E. mundtii, and E. raffinosus. Five clones from E. avium and
E. pseudoavium yielded only a single tuf sequence. These new
sequence data allowed the design of new primers specific for the
enterococcal tufA or tufB sequences. Primers SEQ ID NOs. 543 and
660 were designed to amplify only enterococcal tufA sequences and a
694-bp fragment was amplified from all 17 enterococcal species. The
694-bp sequences of tufA genes from E. columbae, E. malodoratus,
and E. sulfureus were obtained by direct sequencing using these
primers. Primers SEQ ID NOs. 664 and 661 were designed for the
amplification of 730-bp portion of tufB genes and yielded the
expected fragments from 11 enterococcal species, including E.
malodoratus and the 10 enterococcal species in which heterogeneous
tuf sequences were initially found. The sequences of the tufB
fragments for E. avium, E. malodoratus and E. pseudoavium were
determined by direct sequencing using the primers SEQ ID NOs. 664
and 661. Overall, tufA gene fragments were obtained from all 17
enterococcal species but tufB gene fragments were obtained with
only 11 enterococcal species (Table 16).
[0433] The identities between tufA and tufB for each enterococcal
species were 68-79% at the nucleotide level and 81 to 89% at the
amino acid level. The tufA gene is highly conserved among all
enterococcal species with identities varying from 87% to 99% for
DNA and 93% to 99% for amino acid sequences, while the identities
among tufB genes of enterococci varies from 77% to 92% for DNA and
91% to 99% for amino acid sequences, indicating their different
origins and evolution (Table 18). Since E. solitarius has been
transferred to the genus Tetragenococcus, which is also a low G+C
gram-positive bacterium, our sequence comparison did not include
this species as an enterococcus. G+C content of enterococcal tufA
sequences ranged from 40.8% to 43.1%, while that of enterococcal
tufB sequences varied from 37.8% to 46.3%. Based on amino acid
sequence comparison, the enterococcal tufA gene products share
higher identities with those of Abiotrophia adiacens, Bacillus
subtilis, Listeria monocytogenes, S. aureus, and S. epidermidis. On
the other hand, the enterococcal tufB gene products share higher
percentages of amino acid identity with the tuf genes of S.
pneumoniae, S. pyogenes and Lactococcus lactis (Table 18).
[0434] In order to elucidate whether the two enterococcal tuf
sequences encode genuine EF-Tu, the deduced amino acid sequences of
both genes were aligned with other EF-Tu sequences available in
SWISSPROT (Release 38). Sequence alignment demonstrated that both
gene products are highly conserved and carry all conserved residues
present in this portion of prokaryotic EF-Tu (FIG. 4). Therefore,
it appears that both gene products could fulfill the function of
EF-Tu. The partial tuf gene sequences encode the portion of EF-Tu
from residues 117 to 317, numbered as in E. coli. This portion
makes up of the last four .alpha.-helices and two .beta.-strands of
domain I, the entire domain II and the N-terminal part of domain
III on the basis of the determined structures of E. coli EF-Tu.
[0435] Based on the deduced amino acid sequences, the enterococcal
tufB genes have unique conserved residues Lys129, Leu140, Ser230,
and Asp234 (E. coli numbering) that are also conserved in
streptococci and L. lactis, but not in the other bacteria (FIG. 4).
All these residues are located in loops except for Ser230. In other
bacteria the residue Ser230 is substituted for highly conserved
Thr, which is the 5.sup.th residue of the third .beta.-strand of
domain II. This region is partially responsible for the interaction
between the EF-Tu and aminoacyl-tRNA by the formation of a deep
pocket for any of the 20 naturally occurring amino acids. According
to our three-dimensional model (data not illustrated), the
substitution Thr230.fwdarw.Ser in domain II of EF-Tu may have
little impact on the capability of the pocket to accommodate any
amino acid. However, the high conservation of Thr230 comparing to
the unique Ser substitution found only. in streptococci and 11
enterococci could suggest a subtle functional role for this
residue.
[0436] The tuf gene sequences obtained for E. faecalis, S. aureus,
S. pneumoniae and S. pyogenes were compared with their respective
incomplete genome sequence. Contigs with more than 99% identity
were identified. Analysis of the E. faecalis genome data revealed
that the single E. faecalis tuf gene is located within an str
operon where tuf is preceded by fus that encodes the elongation
factor G. This str operon is present in S. aureus and B. subtilis
but not in the two streptococcal genomes examined. The 700-bp or so
sequence upstream the S. pneumoniae tuf gene has no homology with
any known gene sequences. In S. pyogenes, the gene upstream of tuf
is similar to a cell division gene, ftsW, suggesting that the tuf
genes in streptococci are not arranged in a str operon.
[0437] Phylogenetic analysis. Phylogenetic analysis of the tuf
amino acid sequences with representatives of eubacteria,
archeabacteria, and eukaryotes using neighbor-joining and maximum
parsimony methods showed three major clusters representing the
three kingdoms of life. Both methods gave similar topologies
consistent with the rRNA gene data (data not shown). Within the
bacterial clade, the tree is polyphyletic but tufA genes from all
enterococcal species always clustered with those from other low G+C
gram-positive bacteria (except for streptococci and lactococci),
while the tufB genes of the 11 enterococcal species form a distinct
cluster with streptococci and L. lactis (FIG. 5). Duplicated genes
from the same organism do not cluster together, thereby not
suggesting evolution by recent gene duplication.
[0438] Southern hybridization. Southern hybridization of BglII/XbaI
digested genomic DNA from 12 enterococcal species tested with the
tufA probe (DIG-labeled tufA fragment from E. faecium) yielded two
bands of different sizes in 9 species, which also carried two
divergent tuf sequences according to their sequencing data. For E.
faecalis and E. solitarius, a single band was observed indicating
that one tuf gene is present (FIG. 6). A single band was also found
when digested genomic DNA from S. aureus, S. pneumoniae, and S.
pyogenes were hybridized with the tufA probe (data not shown). For
E. faecium, the presence of three bands can be explained by the
existence of a XbaI restriction site in the middle of the tufA
sequence, which was confirmed by sequencing data. Hybridization
with the tufB probe (DIG-labeled tufB fragment of E. faecium)
showed a banding profile similar to the one obtained with the tufA
probe (data not shown).
Discussion
[0439] In this study, we have shown that two divergent copies of
genes encoding the elongation factor Tu are present in some
enterococcal species. Sequence data revealed that both genes are
highly conserved at the amino acid level. One copy (tufA) is
present in all enterococcal species, while the other (tufB) is
present only in 11 of the 17 enterococcal species studied. Based on
16S rRNA sequence analysis, these 11 species are members of three
different enterococcal subgroups (E. avium, E. faecium, and E.
gallinarum species groups) and a distinct species (E. dispar).
Moreover, 16S rDNA phylogeny suggests that these 11 species
possessing 2 tuf genes all share a common ancestor before they
further evolved to become the modern species. Since the six other
species having only one copy diverged from the enterococcal lineage
before that common ancestor, it appears that the presence of one
tuf gene in these six species is not attributable to gene loss.
[0440] Two clusters of low G+C gram-positive bacteria were observed
in the phylogenetic tree of the tuf genes: one contains a majority
of low G+C gram-positive bacteria and the other contains lactococci
and streptococci. This is similar to the finding on the basis of
phylogenetic analysis of the 16S rRNA gene and the hrcA gene coding
for a unique heat-shock regulatory protein. The enterococcal tufA
genes branched with most of the low G+C gram-positive bacteria,
suggesting that they originated from a common ancestor. On the
other hand, the enterococcal tufB genes branched with the genera
Streptococcus and Lactococcus that form a distinct lineage
separated from other low G+C gram-positive bacteria (FIG. 5). The
finding that these EF-Tu proteins share some conserved amino acid
residues unique to this branch also supports the idea that they may
share a common ancestor. Although these conserved residues might
result from convergent evolution upon a specialized function, such
convergence at the sequence level, even for a few residues, seems
to be rare, making it an unlikely event. Moreover, no currently
known selective pressure, if any, would account for keeping one
versus two tuf genes in bacteria. The G+C contents of enterococcal
tufA and tufB sequences are similar, indicating that they both
originated from low G+C gram-positive bacteria, in accordance with
the phylogenetic analysis.
[0441] The tuf genes are present in various copy numbers in
different bacteria. Furthermore, the two tuf genes are normally
associated with characteristic flanking genes. The two tuf gene
copies commonly encountered within gram-negative bacteria are part
of the bacterial str operon and tRNA-tufB operon, respectively. The
arrangement of tufA in the str operon was also found in a variety
of bacteria, including Thermotoga maritima, the most ancient
bacteria sequenced so far, Aquifex aeolicus, cyanobacteria,
Bacillus sp., Micrococcus luteus, Mycobacterium tuberculosis, and
Streptomyces sp. Furthermore, the tRNA-tufB operon has also been
identified in Aquifex aeolicus, Thermus thermophilus, and Chlamydia
trachomatis. The two widespread tuf gene arrangements argue in
favor of their ancient origins. It is noteworthy that most obligate
intracellular parasites, such as Mycoplasma sp., R. prowazekii, B.
burgdorferi, and T. pallidum, contain only one tuf gene. Their
flanking sequences are distinct from the two conserved patterns as
a result of selection for effective propagation by an extensive
reduction in genome size by intragenomic recombination and
rearrangement.
[0442] Most gram-positive bacteria with low G+C content sequenced
to date contain only a single copy of the tuf gene as a part of the
str operon. This is the case for B. subtilis, S. aureus and E.
faecalis. PCR amplification using a primer targeting a conserved
region of the fus gene and the tufA-specific primer SEQ ID NO. 660,
but not the tufB-specific primer SEQ ID NO. 661, yielded the
expected amplicons for all 17 enterococcal species tested,
indicating the presence of the fus-tuf organization in all
enterococci (data not shown). However, in the genomes of S.
pneumoniae and S. pyogenes, the sequences flanking the tuf genes
varies although the tuf gene itself remains highly conserved. The
enterococcal tufB genes are clustered with streptococci, but at
present we do not have enough data to identify the genes flanking
the enterococcal tufB genes. Furthermore, the functional role of
the enterococcal tufB genes remains unknown. One can only postulate
that the two divergent gene copies are expressed under different
conditions.
[0443] The amino acid sequence identities between the enterococcal
tufA and tufB genes are lower than either i) those between the
enterococcal tufA and the tuf genes from other low G+C
gram-positive bacteria (streptococci and lactococci excluded) or
ii) those between the enterococcal tufB and streptococcal and
lactococcal tuf genes. These findings suggest that the enterococcal
tufA genes share a common ancestor with other low G+C gram-positive
bacteria via the simple scheme of vertical evolution, while the
enterococcal tufB genes are more closely related to those of
streptococci and lactococci. The facts that some enterococci
possess an additional tuf gene and that the single streptococcal
tuf gene is nut clustered with other low G+C gram-positive bacteria
cannot be explained by the mechanism of gene duplication or
intrachromosomal recombination. According to sequence and
phylogenetic analysis, we propose that the presence of the
additional copy of the tuf genes in 11 enterococcal species is due
to horizontal gene transfer. The common ancestor of the 11
enterococcal species now carrying tufB genes acquired a tuf gene
from an ancestral streptococcus or a streptococcus-related species
during enterococcal evolution through gene transfer before the
diversification of modern enterococci. Further study of the
flanking regions of the gene may provide more clues for the origin
and function of this gene in enterococci.
[0444] Recent studies of genes and genomes have demonstrated that
considerable horizontal transfer occurred in the evolution of
aminoacyl-tRNA synthetases in all three kingdoms of life. The
heterogeneity of 16S rRNA is also attributable to horizontal gene
transfer in some bacteria, such as Streptomyces, Thermomonospora
chromogena and Mycobacterium celatum. In this study, we provide the
first example in support of a likely horizontal transfer of the tuf
gene encoding the elongation factor Tu. This may be an exception
since stringent functional constraints do not allow for frequent
horizontal transfer of the tuf gene as with other genes. However,
enterococcal tuf genes should not be the only such exception as we
have noticed that the phylogeny of Streptomyces tuf genes is
equally or more complex than that of enterococci. For example, the
three tuf-like genes in a high G+C gram-positive bacterium, S.
ramocissimus, branched with the tuf genes of phylogenetically
divergent groups of bacteria (FIG. 5). Another example may be the
tuf genes in clostridia, which represent a phylogenetically very
broad range of organisms and form a plethora of lines and groups of
various complexities and depths. Four species belonging to three
different clusters within the genus Clostridium have been shown by
Southern hybridization to carry two copies of the tuf gene. Further
sequence data and phylogenetic analysis may help interpreting the
evolution of the elongation factor Tu in these gram-positive
bacteria. Since the tuf genes and 16S rRNA genes are often used for
phylogenetic study, the existence of duplicate genes originating
from horizontal gene transfer may alter the phylogeny of
microorganisms when the laterally acquired copy of the gene is used
for such analysis. Hence, caution should be taken in interpreting
phylogenetic data. In addition, the two tuf genes in enterococci
have evolved separately and are distantly related to each other
phylogenetically. The enterococcal tufB genes are less conserved
and unique to the 11 enterococcal species only. We previously
demonstrated that the enterococcal tufA genes could serve as a
target to develop a DNA-based assay for identification of
enterococci. The enterococcal tufB genes would also be useful in
identification of these 11 enterococcal species.
Example 43
Elongation Factor Tu (tuf) and the F-ATPase Beta-Subunit (atpD) as
Phylogenetic Tools for Species of the Family Enterobacteriaceae
Summary
[0445] The phylogeny of enterobacterial species commonly found in
clinical samples was analyzed by comparing partial sequences of
their elongation factor Tu (tuf) genes and their F-ATPase
beta-subunit (atpD) genes. A 884-bp fragment for tuf and a 884- or
871-bp fragment for atpD were sequenced for 88 strains of 72
species from 25 enterobacterial genera. The atpD sequence analysis
revealed a specific indel to Pantoea and Tatumella species showing
for the first time a tight phylogenetic affiliation between these
two genera. Comprehensive tuf and atpD phylogenetic trees were
constructed and are in agreement with each other. Monophyletic
genera are Yersinia, Pantoea, Edwardsiella, Cedecea, Salmonella,
Serratia, Proteus, and Providencia. Analogous trees were obtained
based on available 16S rDNA sequences from databases. tuf and atpD
phylogenies are in agreement with the 16S rDNA analysis despite the
smaller resolution power for the latter. In fact, distance
comparisons revealed that tuf and atpD genes provide a better
resolution for pairs of species belonging to the family
Enterobacteriaceae. However, 16S rDNA distances are better resolved
for pairs of species belonging to different families. In
conclusion, tuf and atpD conserved genes are sufficiently divergent
to discriminate different species inside the family
Enterobacteriaceae and offer potential for the development of
diagnostic tests based on DNA to identify enterobacterial
species.
Introduction
[0446] Members of the family Enterobacteriaceae are facultatively
anaerobic gram-negative rods, catalase-positive and
oxidase-positive (Brenner, 1984). They are found in soil, water,
plants, and in animals from insects to man. Many enterobacteria are
opportunistic pathogens. In fact, members of this family are
responsible for about 50% of nosocomial infections in the United
States (Brenner, 1984). Therefore, this family is of considerable
clinical importance.
[0447] Major classification studies on the family
Enterobacteriaceae are based on phenotypic traits (Brenner et al.,
1999; Brenner et al., 1980; Dickey & Zumoff, 1988; Farmer III
et al., 1980; Farmer III et al., 1985b; Farmer III et al., 1985a)
such as biochemical reactions and physiological characteristics.
However, phenotypically distinct strains may be closely related by
genotypic criteria and may belong to the same genospecies
(Bercovier et al., 1980; Hartl & Dykhuizen, 1984). Also,
phenotypically close strains (biogroups) may belong to different
genospecies, like Klebsiella pneumoniae and Enterobacter aerogenes
(Brenner, 1984) for example. Consequently, identification and
classification of certain species may be ambiguous with techniques
based on phenotypic tests (Janda et al., 1999; Kitch et al., 1994;
Sharma et al., 1990).
[0448] More advances in the classification of members of the family
Enterobacteriaceae have come from DNA-DNA hybridization studies
(Brenner et al., 1993; Brenner et al., 1986; Brenner, et al., 1980;
Farmer III, et al., 1980; Farmer III, et al., 1985b; Izard et al.,
1981; Steigerwalt et al., 1976). Furthermore, the phylogenetic
significance of bacterial classification based on 16S rDNA
sequences has been recognized by many workers (Stackebrandt &
Goebel, 1994; Wayne et al., 1987). However, members of the family
Enterobacteriaceae have not been subjected to extensive
phylogenetic analysis of 16S rDNA (Sproer et al., 1999). In fact,
this molecule was not thought to solve taxonomic problems
concerning closely related species because of its very high degree
of conservation (Brenner, 1992; Sproer, et al., 1999). Another
drawback of the 16S rDNA gene is that it is found in several copies
within the genome (seven in Escherichia coli and Salmonella
typhimurium) (Hill & Harnish, 1981). Due to sequence divergence
between the gene copies, direct sequencing of PCR products is often
not suitable to achieve a representative sequence (Cilia et al.,
1996; Hill & Harnish, 1981). Other genes such as gap and ompA
(Lawrence et al., 1991), rpoB (Mollet et al., 1997), and infB
(Hedegaard et al., 1999) were used to resolve the phylogeny of
enterobacteria. However, none of these studies covered an extensive
number of species.
[0449] tuf and atpD are the genes encoding the elongation factor Tu
(EF-Tu) and the F-ATPase beta-subunit, respectively. EF-Tu is
involved in peptide chain formation (Ludwig et al., 1990). The two
copies of the tuf gene (tufA and tufB) found in enterobacteria
(Sela et al., 1989) share high identity level (99%) in Salmonella
typhimurium and in E. coli. The recombination phenomenon could
explain sequence homogenization between the two copies (Abdulkarim
& Hughes, 1996; Grunberg-Manago, 1996). F-ATPase is present on
the plasma membranes of eubacteria (Nelson & Taiz, 1989). It
functions mainly in ATP synthesis (Nelson & Taiz, 1989) and the
beta-subunit contains the catalytic site of the enzyme. EF-Tu and
F-ATPase are highly conserved throughout evolution and shows
functional constancy (Amann et al., 1988; Ludwig, et al., 1990).
Recently, phylogenies based on protein sequences from EF-Tu and
F-ATPase beta-subunit showed good agreement with each other and
with the rDNA data (Ludwig et al., 1993).
[0450] We elected to sequence 884-bp fragments of tuf and atpD from
88 clinically relevant enterobacterial strains representing 72
species from 25 genera. These sequences were used to create
phylogenetic trees that were compared with 16S rDNA trees. These
trees revealed good agreement with each others and demonstrated the
high resolution of tuf and atpD phylogenies at the species
level.
Materials and Methods
[0451] Bacterial strains and genomic material. All bacterial
strains used in this study were obtained from the American Type
Culture Collection (ATCC) or the Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (DSMZ). These enterobacteria
can all be recovered from clinical specimens, but not all are
pathogens. Whenever possible, we choose type strains.
Identification of all strains was confirmed by classical
biochemical tests using the automated system MicroScan WalkAway-96
system equipped with a Negative BP Combo Panel Type 15 (Dade
Behring Canada). Genomic DNA was purified using the G NOME DNA kit
(Bio 101). Genomic DNA from Yersinia pestis was kindly provided by
Dr. Robert R. Brubaker. Strains used in this study and their
descriptions are shown in Table 19.
[0452] PCR primers. The eubacterial tuf and atpD gene sequences
available from public databases were analyzed using the GCG package
(version 8.0) (Genetics Computer Group). Based on multiple sequence
alignments, two highly conserved regions were chosen for each
genes, and PCR primers were derived from these regions with the
help of Oligo primer analysis software (version 5.0) (National
Biosciences). A second 5' primer was design to amplify the gene
atpD for few enterobacteria difficult to amplify with the first
primer set. When required, the primers contained inosines or
degeneracies to account for variable positions. Oligonucleotide
primers were synthesized with a model 394 DNA/RNA synthesizer (PE
Applied Biosystems). PCR primers used in this study are listed in
Table 20.
[0453] DNA sequencing. An 884-bp portion of the tuf gene and an
884-bp portion (or alternatively an 871-bp portion for a few
enterobacterial strains) of the atpD gene were sequenced for all
enterobacteria listed in the first strain column of Table 19.
Amplification was performed with 4 ng of genomic DNA. The 40-.mu.l
PCR mixtures used to generate PCR products for sequencing contained
1.0 .mu.M each primer, 200 .mu.M each deoxyribonucleoside
triphosphate (Pharmacia Biotech), 10 mM Tris-HCl (pH 9.0 at
25.degree. C.), 50 mM KCl, 0.1% (w/v) Triton X-100, 2.5 mM
MgCl.sub.2, 0.05 mM BSA, 0.3 U of Taq DNA polymerase (Promega)
coupled with TaqStart.TM. antibody (Clontech Laboratories). The
TaqStart.TM. neutralizing monoclonal antibody for Taq DNA
polymerase was added to all PCR mixtures to enhance efficiency of
amplification (Kellogg et al., 1994). The PCR mixtures were
subjected to thermal cycling (3 min at 95.degree. C. and then 35
cycles of 1 min at 95.degree. C., 1 min at 55.degree. C. for tuf or
50.degree. C. for atpD, and 1 min at 72.degree. C., with a 7-min
final extension at 72.degree. C.) using a PTC-200 DNA Engine
thermocycler (MJ Research). PCR products having the predicted sizes
were recovered from an agarose gel stained for 15 min with 0.02% of
methylene blue followed by washing in sterile distilled water for
15 min twice (Flores et al., 1992). Subsequently, PCR products
having the predicted sizes were recovered from gels using the
QIAquick gel extraction kit (QIAGEN).
[0454] Both strands of the purified amplicons were sequenced using
the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit
(PE Applied Biosystems) on an automated DNA sequencer (Model 377).
Amplicons from two independent PCR amplifications sequenced for
each strain to ensure the absence of sequencing errors attributable
to nucleotide miscorporations by the Taq DNA polymerase. Sequence
assembly was performed with the aid of Sequencher 3.0 software
(Gene Codes).
[0455] Phylogenetic analysis. Multiple sequence alignments were
performed using PileUp from the GCG package (Version 10.0)
(Genetics Computer Group) and checked by eye with the editor SeqLab
to edit sequences if necessary and to note which regions were to be
excluded for phylogenetic analysis. Vibrio cholerae and Shewanella
putrefaciens were used as outgroups. Bootstrap subsets (750 sets)
and phylogenetic trees were generated with the Neighbor Joining
algorithm from Dr. David Swofford's PAUP (Phylogenetic Analysis
Using Parsimony) Software version 4.0b4 (Sinauer Associates) and
with tree-bisection branch-swapping. The distance model used was
Kimura (1980) two-parameter. Relative rate test was performed with
the aid of Phyltest program version 2.0 (c).
Results and Discussion
DNA Amplification, Sequencing And Sequence Alignments
[0456] A PCR product of the expected size of 884 by was obtained
for tuf and of 884 or 871 by for atpD from all bacterial strains
tested. After subtracting for biased primer regions and ambiguous
single strand data, sequences of at least 721 by for tuf and 713 by
for atpD were submitted to phylogenetic analyses. These sequences
were aligned with tuf and atpD sequences available in databases to
verify that the nucleotide sequences indeed encoded a part of
tested genes. Gaps were excluded to perform phylogenetic
analysis.
Signature Sequences
[0457] From the sequence alignments obtained from both tested
genes, only one insertion was observed. This five amino acids
insertion is located between the positions 325 and 326 of atpD gene
of E. coli strain K-12 (Saraste et al., 1981) and can be considered
a signature sequence of Tatumella ptyseos and Pantoea species (FIG.
7). The presence of a conserved indel of defined length and
sequence and flanked by conserved regions could suggest a common
ancestor, particularly when members of a given taxa share this
indel (Gupta, 1998). To our knowledge, high relatedness between the
genera Tatumella and Pantoea is demonstrated for the first
time.
[0458] Enterobacter agglomerans ATCC 27989 sequence does not
possess the five amino acid indel (FIG. 7). This indel could
represent a useful marker to help resolve the Enterobacter
agglomerans and Pantoea classification. Indeed, the transfer of
Enterobacter agglomerans to Pantoea agglomerans was proposed in
1989 by Gavini et al. (Gavini et al., 1989). However, some strains
are provisionally classified as Pantoea sp. until their
interrelatedness is elucidated (Gavini, et al., 1989). Since the
transfer was proposed, the change of nomenclature has not yet been
made for all Enterobacter agglomerans in the ATCC database. The
absence of the five amino acids indel suggests that some strains of
Enterobacter agglomerans most likely do not belong to the genus
Pantoea.
Phylogenetic Trees Based on Partial Tuf Sequences, Atpd Sequences,
and Published 16S Rdna Data of Members of the
Enterobacteriaceae.
[0459] Representative trees constructed from tuf and atpD sequences
with the neighbor-joining method are shown in FIG. 8. The
phylogenetic trees generated from partial tuf sequences and atpD
sequences are very similar. Nevertheless, atpD tree shows more
monophyletic groups corresponding to species that belong to the
same genus. These groups are more consistent with the actual
taxonomy. For both genes, some genera are not monophyletic. These
results support previous phylogenies based on the genes gap and
ompA (Lawrence, et al., 1991), rpoB (Mollet, et al., 1997), and
infB (Hedegaard, et al., 1999) which all showed that the genera
Escherichia and Klebsiella are polyphyletic. There were few
differences in branching between tuf and atpD genes.
[0460] Even though Pantoea agglomerans and Pantoea dispersa indels
were excluded for phylogenetic analysis, these two species grouped
together and were distant from Enterobacter agglomerans ATCC 27989,
adding another evidence that the latter species is heterogenous and
that not all members of this species belong to the genus Pantoea.
In fact, the E. agglomerans strain ATCC 27989 exhibits branch
lengths similar to others Enterobacter species with both genes.
Therefore, we suggest that this strain belong to the genus
Enterobacter until further reclassification of that genus.
[0461] tuf and atpD trees exhibit very short genetic distances
between taxa belonging to the same genetic species including
species segregated for clinical considerations. This first concern
E. coli and Shigella species that were confirmed to be the same
genetic species by hybridization studies (Brenner et al., 1972;
Brenner et al., 1972; Brenner et al., 1982) and phylogenies based
on 16S rDNA (Wang et al., 1997) and rpoB genes (Mollet, et al.,
1997). Hybridization studies (Bercovier, et al., 1980) and
phylogeny based on 16S rDNA genes (Ibrahim et al., 1994)
demonstrated also that Yersinia pestis and Y. pseudotuberculosis
are the same genetic species. Among Yersinia pestis and Y.
pseudotuberculosis, the three Klebsiella pneumoniae subspecies, E.
coli-Shigella species, and Salmonella choleraesuis subspecies,
Salmonella is a less tightly knit species than the other genetic
species. The same is true for E. coli and Shigella species.
[0462] Escherichia fergusonii is very close to E. coli-Shigella
genetic species. This observation is corroborated by 16S rDNA
phylogeny (McLaughlin et al., 2000) but not by DNA hybridization
values. In fact, E. fergusonii is only 49% to 63% related to E.
coli-Shigella (Farmer III, et al., 1985b). It was previously
observed that very recently diverged species may not be
recognizable based on 16S rDNA sequences although DNA hybridization
established them as different species (Fox et al., 1992).
Therefore, E. fergusonii could be a new "quasi-species".
[0463] atpD phylogeny revealed Salmonella subspecies divisions
consistent with the actual taxonomy. This result was already
observed by Christensen et al. (Christensen & Olsen, 1998).
Nevertheless, tuf partial sequences discriminate less than atpD
between Salmonella subspecies.
[0464] Overall, tuf and atpD phylogenies exhibit enough divergence
between species to ensure efficient discrimination. Therefore, it
could be easy to distinguish phenotypically close enterobacteria
belonging to different genetic species such as Klebsiella
pneumoniae and Enterobacter aerogenes.
[0465] Phylogenetic relationships between Salmonella, E. coli and
C. freundii are not well defined. 16S rDNA and 23S rDNA sequence
data reveals a closer relationship between Salmonella and E. coli
than between Salmonella and C. freundii (Christensen et al., 1998),
while DNA homology studies (Selander et al., 1996) and infB
phylogeny (Hedegaard, et al., 1999) showed that Salmonella is more
closely related to C. freundii than to E. coli. In that regard, tuf
and atpD phylogenies are coherent with 16S rDNA and 23S rDNA
sequence analysis.
[0466] Phylogenetic analyses were also performed using amino acids
sequences. tuf tree based on amino acids is characterized by a
better resolution between taxa outgroup and taxa ingroup
(enterobacteria) than tree based on nucleic acids whereas atpD
trees based on amino acids and nucleic acids give almost the same
resolution between taxa outgroup and ingroup (data not shown).
[0467] Relative rate test (or two cluster test (Takezaki et al.,
1995)) evaluates if evolution is constant between two taxa. Before
to apply the test, the topology of a tree is determined by
tree-building method without the assumption of rate constancy.
Therefore, two taxa (or two groups of taxa) are compared with a
third taxon that is an outgroup of the first two taxa (Takezaki, et
al., 1995). Few pairs of taxa that exhibited a great difference
between their branch lengths at particular nodes were chosen to
perform the test. This test reveals that tuf and atpD are not
constant in their evolution within the family Enterobacteriaceae.
For tuf, for example, the hypothesis of rate constancy is rejected
(Z value higher than 1.96) between Yersinia species. The same is
true for Proteus species. For atpD, for example, evolution is not
constant between Proteus species, between Proteus species and
Providencia species, and between Yersinia species and Escherichia
coli. For 16S rDNA, for example, evolution is not constant between
two E. coli, between E. coli and Enterobacter aerogenes, and
between E. coli and Proteus vulgaris. These results suggest that
tuf, atpD and 16S rDNA could not serve as a molecular clock for the
entire family Enterobacteriaceae.
[0468] Since the number and the nature of taxa can influence
topology of trees, phylogenetic trees from tuf and atpD were
reconstructed using sequences corresponding to strains for which
16S rDNA genes were published in GenEMBL. These trees were similar
to those generated using 16S rDNA (FIG. 9). Nevertheless, 16S rDNA
tree gave poorer resolution power than tuf and atpD gene trees.
Indeed, these latter exhibited less multifurcation (polytomy) than
the 16S rDNA tree.
Comparison of Distances Based on tuf, atpD, and 16S rDNA data.
[0469] tuf, atpD, and 16S rDNA distances (i.e. the number of
differences per nucleotide site) were compared with each other for
each pair of strains. We found that the tuf and atpD distances were
respectively 2.268.+-.0.965 and 2.927.+-.0.896 times larger than
16S rDNA distances (FIGS. 10a and b). atpD distances were
1.445.+-.0.570 times larger than tuf distances (FIG. 10c). FIG. 10
also shows that the tuf, atpD, and 16S rDNA distances between
members of different species of the same genus (0.053.+-.0.034,
0.060.+-.0.020, and 0.024.+-.0.010, respectively) were in mean
smaller than the distances between members of different genera
belonging to the same family (0.103.+-.0.053, 0.129.+-.0.051, and
0.044.+-.0.013, respectively). However, the overlap exhibits with
standard deviations add to a focus of evidences that some
enterobacterial genera are not well defined (Brenner, 1984). In
fact, many distances for pairs of species especially belonging to
the genera Escherichia, Shigella, Enterobacter, Citrobacter,
Klebsiella, and Kluyvera overlap distances for pairs of species
belonging to the same genus (FIG. 10). For example, distances for
pairs composed by species of Citrobacter and species of Klebsiella
overlap distances for pairs composed by two Citrobacter or by two
Klebsiella.
[0470] Observing the distance distributions, 16S rDNA distances
reveal a clear separation between the families Enterobacteriaceae
and Vibrionaceae despite the fact that the family Vibrionaceae is
genetically very close to the Enterobacteriaceae (FIGS. 10a and b).
Nevertheless, tuf and atpD show higher discriminating power below
the family level (FIGS. 10a and b).
[0471] There were some discrepancies in the relative distances for
the same pairs of taxa between the two genes studied. First,
distances between Yersinia species are at least two times lower for
atpD than for tuf (FIG. 10c). Also, distances at the family level
(between Enterobacteriaceae and Vibrionaceae) show that
Enterobacteriaceae is a tightlier knit family with atpD gene
(Proteus genus excepted) than with tuf gene. Both genes well
delineate taxa belonging to the same species. There is one
exception with atpD: Klebsiella planticola and K. ornithinolithica
belong to the same genus but fit with taxa belonging to the same
species (FIGS. 10a and c). These two species are also very close
genotypically with tuf gene. This suggest that Klebsiella
planticola and K. ornithinolithica could be two newborn species.
tuf and atpD genes exhibit little distances between Escherichia
fergusonii and E. coli-Shigella species. Unfortunately, comparison
with 16S rDNA could not be achieved because the E. fergusonii 16S
rDNA sequence is not yet accessible in GenEMBL database. Therefore,
the majority of phenotypically close enterobacteria could be easily
discriminated genotypically using tuf and atpD gene sequences.
[0472] In conclusion, tuf and atpD genes exhibit phylogenies
consistent with 16S rDNA genes phylogeny. For example, they reveal
that the family Enterobacteriaceae is monophyletic. Moreover, tuf
and atpD distances provide a higher discriminating power than 16S
rDNA distances. In fact, tuf and atpD genes discriminate well
between different genospecies and are conserved between strains of
the same genetic species in such a way that primers and molecular
probes for diagnostic purposes could be designed. Preliminary
studies support these observations and diagnostic tests based on
tuf and atpD sequence data to identify enterobacteria are currently
under development.
Example 44
Testing New Pairs of PCR Primers Selected from Two Species-Specific
Genomic DNA Fragments which are Objects of Our Assigned U.S. Pat.
No. 6,001,564
[0473] Objective. The goal of these experiments is to demonstrate
that it is relatively easy for a person skilled in the art to find
other PCR primer pairs from the species-specific fragments used as
targets for detection and identification of a variety of
microorganisms. In fact, we wish to prove that the PCR primers
previously tested by our group and which are objects of the present
patent application are not the only possible good choices for
diagnostic purposes. For this example, we used diagnostic targets
described in our assigned U.S. Pat. No. 6,001,564.
[0474] Experimental strategy. We have selected randomly two
species-specific genomic DNA fragments for this experiment. The
first one is the 705-bp fragment specific to Staphylococcus
epidermidis (SEQ ID NO: 36 from U.S. Pat. No. 6,001,564) while the
second one is the 466-bp fragment specific to Moraxella catarrhalis
(SEQ ID NO: 29 from U.S. Pat. No. 6,001,564). Subsequently, we have
selected from these two fragments a number of PCR primer pairs
other than those previously tested. We have chosen 5 new primer
pairs from each of these two sequences which are well dispersed
along the DNA fragment (FIGS. 11 and 12). We have tested these
primers for their specificity and compared them with the original
primers previously tested. For the specificity tests, we have
tested all bacterial species closely related to the target species
based on phylogenetic analysis with three conserved genes (rRNA
genes, tuf and atpD). The rational for selecting a restricted
number of bacterial species to evaluate the specificity of the new
primer pairs is based on the fact that the lack of specificity of a
DNA-based assay is attributable to the detection of closely related
species which are more similar at the nucleotide level. Based on
the phylogenetic analysis, we have selected (i) species from the
closely related genus Staphylococcus, Enterococcus, Streptococcus
and Listeria to test the specificity of the S. epidermidis-specific
PCR assays and (ii) species from the closely related genus
Moraxella, Kingella and Neisseria to test the specificity of the M.
catarrhalis-specific PCR assays.
Materials and Methods
[0475] Bacterial strains. All bacterial strains used for these
experiments were obtained from the American Type Culture Collection
(ATCC, Rockville, Md.).
[0476] Genomic DNA isolation. Genomic DNA was purified from the
ATCC reference strains by using the G-nome DNA kit (Bio 101 Inc.,
Vista, Calif.).
[0477] Oligonucleotide design and synthesis. PCR primers were
designed with the help of the Oligo.TM. primer analysis software
Version 4.0 (National Biosciences Inc., Plymouth, Minn.) and
synthesized using a model 391 DNA synthesizer (Applied Biosystems,
Foster City, Calif.).
[0478] PCR assays. All PCR assays were performed by using genomic
DNA purified from reference strains obtained from the ATCC. One
.mu.l of purified DNA preparation (containing 0.01 to 1 ng of DNA
per .mu.l) was added directly into the PCR reaction mixture. The 20
.mu.L PCR reactions contained final concentrations of 50 mM KCl, 10
mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl.sub.2, 0.4
.mu.M of each primer, 200 .mu.M of each of the four dNTPs and 0.5
unit of Taq DNA polymerase (Promega, Madison, Wis.) combined with
the TaqStart.TM. antibody (Clontech Laboratories Inc., Palo Alto,
Calif.). An internal control was integrated into all amplification
reactions to verify the efficiency of the amplification reaction as
well as to ensure that significant PCR inhibition was absent.
Primers amplifying a region of 252 by from a control plasmid added
to each amplification reaction were used to provide the internal
control. PCR reactions were then subjected to thermal cycling (3
min at 95.degree. C. followed by 30 cycles of 1 second at
95.degree. C. for the denaturation step and 30 seconds at 50 to
65.degree. C. for the annealing-extension step) using a PTC-200
thermal cycler (MJ Research Inc., Watertown, Mass.). PCR
amplification products were then analyzed by standard agarose gel
(2%) electrophoresis. Amplification products were visualized in
agarose gels containing 0.25 .mu.g/mL of ethidium bromide under UV
at 254 nm.
Results
[0479] Tables 21 and 22 show the results of specificity tests with
the 5 new primer pairs selected from SEQ ID NO: 29 (specific to M.
catarrhalis from U.S. Pat. No. 6,001,564) and SEQ ID NO: 36
(specific to S. epidermidis from U.S. Pat. No. 6,001,564),
respectively. In order to evaluate the performance of these new
primers pairs, we compared them in parallel with the original
primer pairs previously tested.
[0480] For M. catarrhalis, all of the 5 selected PCR primer pairs
were specific for the target species because none of the closely
related species could be amplified (Table 21). In fact, the
comparison with the original primer pair SEQ ID NO: 118+SEQ ID NO:
119 (from U.S. Pat. No. 6,001,564) revealed that all new pairs
showed identical results in terms of specificity and sensitivity
thereby suggesting their suitability for diagnostic purposes.
[0481] For S. epidermidis, 4 of the 5 selected PCR primer pairs
were specific for the target species (Table 22). It should be noted
that for 3 of these four primer pairs the annealing temperature had
to be increased from 55.degree. C. to 60 or 65.degree. C. to attain
specificity for S. epidermidis. Again the comparison with the
original primer pair SEQ ID NO: 145+SEQ ID NO: 146 (from U.S. Pat.
No. 6,001,564) revealed that these four primer pairs were as good
as the original pair. Increasing the annealing temperature for the
PCR amplification is well known by persons skilled in the art to be
a very effective way to improve the specificity of a PCR assay
(Persing et al., 1993, Diagnostic Molecular Microbiology:
Principles and Applications, American Society for Microbiology,
Washington, D.C.; Ehrlich and Greenberg, 1994, PCR-based
Diagnostics in Infectious Disease, Blackwell Scientific
Publications, Boston, Mass.). In fact, those skilled in the art are
well aware of the fact that the annealing temperature is critical
for the optimization of PCR assays. Only the primer pair
VBsep3+VBsep4 amplified bacterial species other than S. epidermidis
including the staphylococcal species S. capitis, S. cohnii, S.
aureus, S. haemolyticus and S. hominis (Table 22). For this
non-specific primer pair, increasing the annealing temperature from
55 to 65.degree. C. was not sufficient to attain the desired
specificity. One possible explanation for the fact that it appears
slightly easier to select species-specific primers for M
catarrhalis than for S. epidermidis is that M catarrhalis is more
isolated in phylogenetic trees than S. epidermidis. The large
number of coagulase negative staphylococcal species such as S.
epidermidis is largely responsible for this phylogenetic
clustering.
Conclusion
[0482] These experiment clearly show that it is relatively easy for
a person skilled in the art to select, from the species-specific
DNA fragments selected as target for identification, PCR primer
pairs suitable for diagnostic purposes other than those previously
tested. The amplification conditions can be optimize by modifying
critical variables such as the annealing temperature to attain the
desired specificity and sensitivity. Consequently, we consider that
it is legitimate to claim any possible primer sequences selected
from the species-specific fragment and that it would be unfair to
grant only the claims dealing with the primer pairs previously
tested. By extrapolation, these results strongly suggest that it is
also relatively easy for a person skilled in the art to select,
from the species-specific DNA fragments, DNA probes suitable for
diagnostic purposes other than those previously tested.
Example 45
Testing Modified Versions of PCR Primers Derived from the Sequence
of Several Primers which are Objects of U.S. Pat. No.
6,001,564.
[0483] Objective. The purpose of this project is to verify the
efficiency of amplification by modified PCR primers derived from
primers previously tested. The types of primer modifications to be
tested include (i) variation of the sequence at one or more
nucleotide positions and (ii) increasing or reducing the length of
the primers. For this example, we used diagnostic targets described
in U.S. Pat. No. 6,001,564.
Experimental Strategy:
[0484] Testing Primers with Nucleotide Changes
[0485] We have designed 13 new primers which are derived from the
S. epidermidis-specific SEQ ID NO: 146 from U.S. Pat. No. 6,001,564
(Table 23). These primers have been modified at one or more
nucleotide positions. As shown in Table 23, the nucleotide changes
were introduced all along the primer sequence. Furthermore, instead
of modifying the primer at any nucleotide position, the nucleotide
changes were introduced at the third position of each codon to
better reflect potential genetic variations in vivo. It should be
noted that no nucleotide changes were introduced at the 3' end of
the oligonucleotide primers because those skilled in the art are
well aware of the fact that mismatches at the 3' end should be
avoided (Persing et al., 1993, Diagnostic Molecular Microbiology:
Principles and Applications, American Society for Microbiology,
Washington, D.C.). All of these modified primers were tested in PCR
assays in combination with SEQ ID NO: 145 from U.S. Pat. No.
6,001,564 and the efficiency of the amplification was compared with
the original primer pair SEQ ID NO: 145+SEQ ID NO: 146 previously
tested in U.S. Pat. No. 6,001,564.
Testing Shorter or Longer Versions of Primers
[0486] We have designed shorter and longer versions of the original
S. epidermidis-specific PCR primer pair SEQ ID NO: 145+146 from
U.S. Pat. No. 6,001,564 (Table 24) as well as shorter versions of
the original P. aeruginosa-specific primer pair SEQ ID NO: 83+84
from U.S. Pat. No. 6,001,564 (Table 25). As shown in Tables 24 and
25, both primers of each pair were shortened or lengthen to the
same length. Again, those skilled in the art know that the melting
temperature of both primers from a pair should be similar to avoid
preferential binding at one primer binding site which is
detrimental in PCR (Persing et al., 1993, Diagnostic Molecular
Microbiology: Principles and Applications, American Society for
Microbiology, Washington, D.C.; Ehrlich and Greenberg, 1994,
PCR-based Diagnostics in Infectious Disease, Blackwell Scientific
Publications, Boston, Mass.). All of these shorter or longer primer
versions were tested in PCR assays and the efficiency of the
amplification was compared with the original primer pair SEQ ID NOs
145 and 146.
Materials and Methods
[0487] See the Materials and methods section of Example 44.
Results
[0488] Testing Primers with Nucleotide Changes
[0489] The results of the PCR assays with the 13 modified versions
of SEQ ID NO: 146 from U.S. Pat. No. 6,001,564 are shown in Table
23. The 8 modified primers having a single nucleotide variation
showed an efficiency of amplification identical to the original
primer pair based on testing with 3 different dilutions of genomic
DNA. The four primers having two nucleotide variations and primer
VBmut12 having 3 nucleotide changes also showed PCR results
identical to those obtained with the original pair. Finally, primer
VBmut13 with four nucleotide changes showed a reduction in
sensitivity by approximately one log as compared with the original
primer pair. However, reducing the annealing temperature from 55 to
50.degree. C. gave an efficiency of amplification very similar to
that observed with the original primer pair (Table 23). In fact,
reducing the annealing temperature of PCR cycles represents an
effective way to reduce the stringency of hybridization for the
primers and consequently allows the binding of probes with
mismatches (Persing et al., 1993, Diagnostic Molecular
Microbiology: Principles and Applications, American Society for
Microbiology, Washington, D.C.). Subsequently, we have confirmed
the specificity of the PCR assays with each of these 13 modified
versions of SEQ ID NO: 146 from U.S. Pat. No. 6,001,564 by
performing amplifications from all bacterial species closely
related to S. epidermidis which are listed in Table 22.
Testing Shorter or Longer Versions of Primers
[0490] For these experiments, two primer pairs were selected: i)
SEQ ID NO: 145+146 from U.S. Pat. No. 6,001,564 (specific to S.
epidermidis) which are AT rich and ii) SEQ ID NO: 83+84 (specific
to P. aeruginosa) which are GC rich. For the AT rich sequence,
primers of 15 to 30 nucleotide in length were designed (Table 24)
while for the GC rich sequences, primers of 13 to 19 nucleotide in
length were designed (Table 25).
[0491] Table 24 shows that, for an annealing temperature of
55.degree. C., the 30-25-, 20- and 17-nucleotide versions of SEQ ID
NO: 145 and 146 from U.S. Pat. No. 6,001,564 all showed identical
results as compared with the original primer pair except that the
17-nucleotide version amplified slightly less efficiently the S.
epidermidis DNA. Reducing the annealing temperature from 55 to
45.degree. C. for the 17-nucleotide version allowed to increase the
amplification efficiency to a level very similar to that with the
original primer pair (SEQ ID NO: 145+146 from U.S. Pat. No.
6,001,564). Regarding the 15-nucleotide version, there was
amplification of S. epidermidis DNA only when the annealing
temperature was reduced to 45.degree. C. Under those PCR conditions
the assay remained S. epidermidis-specific but the amplification
signal with S. epidermidis DNA was slightly lower as compared with
the original primer pair. Subsequently, we have further confirmed
the specificity of the shorter or longer versions by amplifying DNA
from all bacterial species closely related to S. epidermidis which
are listed in Table 22.
[0492] Table 25 shows that, for an annealing temperature of
55.degree. C., all shorter versions of SEQ ID NO: 83 and 84 from
U.S. Pat. No. 6,001,564 showed identical PCR results as compared
with the original primer pair. As expected, these results show that
it is simpler to reduce the length of GC rich as compared with AT
rich. This is attributable to the fact that GC binding is more
stable than AT binding.
Conclusion
[0493] Testing Primers with Nucleotide Changes
[0494] The above experiments clearly show that PCR primers may be
modified at one or more nucleotide positions without affecting the
specificity and the sensitivity of the PCR assay. These results
strongly suggest that a given oligonucleotide can detect variant
genomic sequences from the target species. In fact, the nucleotide
changes in the selected primers were purposely introduced at the
third position of each codon to mimic nucleotide variation in
genomic DNA. Thus we conclude that it is justified to claim "a
variant thereof" for i) the SEQ IDs of the fragments and
oligonucleotides which are object of the present patent application
and ii) genomic variants of the target species.
Testing Shorter or Longer Versions of Primers
[0495] The above experiments clearly show that PCR primers may be
shorter or longer without affecting the specificity and the
sensitivity of the PCR assay. We have showed that oligonucleotides
ranging in sizes from 13 to 30 nucleotides may be as specific and
sensitive as the original primer pair from which they were derived.
Consequently, these results suggest that it is not exaggerated to
claim sequences having at least 12 nucleotide in length.
[0496] This invention has been described herein above, and it is
readily apparent that modifications can be made thereto without
departing from the spirit of this invention. These modifications
are under the scope of this invention, as defined in the appended
claims.
TABLE-US-00001 TABLE 1 Distribution (%) of nosocomial pathogens for
various human infections in USA (1990-1992).sup.1. Pathogen
UTI.sup.2 SSI.sup.3 BSI.sup.4 Pneumonia CSF.sup.5 Escherichia coli
27 9 5 4 2 Staphylococcus aureus 2 21 17 21 2 Staphylococcus
epidermidis 2 6 20 0 1 Enterococcus faecalis 16 12 9 2 0
Enterococcus faecium 1 1 0 0 0 Pseudomonas aeruginosa 12 9 3 18 0
Klebsiella pneumoniae 7 3 4 9 0 Proteus mirabilis 5 3 1 2 0
Streptococcus pneumoniae 0 0 3 1 18 Group B Streptococci 1 1 2 1 6
Other streptococci 3 5 2 1 3 Haemophilus influenzae 0 0 0 6 45
Neisseria meningitidis 0 0 0 0 14 Listeria monocytogenes 0 0 0 0 3
Other enterococci 1 1 0 0 0 Other staphylococci 2 8 13 2 0 Candida
albicans 9 3 5 5 0 Other Candida 2 1 3 1 0 Enterobacter sp. 5 7 4
12 2 Acinetobacter sp. 1 1 2 4 2 Citrobacter sp. 2 1 1 1 0 Serratia
marcescens 1 1 1 3 1 Other Klebsiella 1 1 1 2 1 Others 0 6 4 5 0
.sup.1Data recorded by the National Nosocomial Infections
Surveillance (NNIS) from 80 hospitals (Emori and Gaynes, 1993,
Clin. Microbiol. Rev., 6: 428-442). .sup.2Urinary tract infection.
.sup.3Surgical site infection. .sup.4Bloodstream infection.
.sup.5Cerebrospinal fluid.
TABLE-US-00002 TABLE 2 Distribution (%) of bloodstream infection
pathogens in Quebec (1995), Canada (1992), UK (1969-1988) and USA
(1990-1992). UK.sup.3 Community- Hospital- USA.sup.4 Organism
Quebec.sup.1 Canada.sup.2 acquired acquired Hospital-acquired E.
coli 15.6 53.8 24.8 20.3 5.0 S. epidermidis and 25.8 -- 0.5 7.2
31.0 other CoNS.sup.5 S. aureus 9.6 -- 9.7 19.4 16.0 S. pneumoniae
6.3 -- 22.5 2.2 -- E. faecalis 3.0 -- 1.0 4.2 -- E. faecium 2.6 --
0.2 0.5 -- Enterococcus sp. -- -- -- -- 9.0 H. influenzae 1.5 --
3.4 0.4 -- P. aeruginosa 1.5 8.2 1.0 8.2 3.0 K. pneumoniae 3.0 11.2
3.0 9.2 4.0 P. mirabilis -- 3.9 2.8 5.3 1.0 S. pyogenes -- -- 1.9
0.9 -- Enterobacter sp. 4.1 5.5 0.5 2.3 4.0 Candida sp. 8.5 -- --
1.0 8.0 Others 18.5 17.4 28.7 18.9 19.0 .sup.1Data obtained for 270
isolates collected at the Centre Hospitalier del'Universite Laval
(CHUL) during a 5 month period (May to October 1995). .sup.2Data
from 10 hospitals throughout Canada representing 941 gram-negative
isolates. (Chamberland et al., 1992, Clin. Infect. Dis., 15:
615-628). .sup.3Data from a 20-year study (1969-1988) for nearly
4000 isolates. (Eykyn et al., 1990, J. Antimicrob. Chemother.,
Suppl. C, 25: 41-58). .sup.4Data recorded by the National
Nosocomial Infections Surveillance (NNIS) from 80 hospitals (Emori
and Gaynes, 1993, Clin. Microbiol. Rev., 6: 428-442).
.sup.5Coagulase-negative staphylococci.
TABLE-US-00003 TABLE 3 Distribution of positive and negative
clinical specimens tested at the microbiology laboratory of the
CHUL (February 1994-January 1995). % Clinical specimens No. of
samples % of positive of negative and/or sites tested (%) specimens
specimens Urine 17,981 (54.5) 19.4 80.6 Blood culture/marrow 10,010
(30.4) 6.9 93.1 Sputum 1,266 (3.8) 68.4 31.6 Superficial pus 1,136
(3.5) 72.3 27.7 Cerebrospinal fluid 553 (1.7) 1.0 99.0 Synovial
fluid 523 (1.6) 2.7 97.3 Respiratory tract 502 (1.5) 56.6 43.4 Deep
pus 473 (1.4) 56.8 43.2 Ears 289 (0.9) 47.1 52.9 Pleural and
pericardial fluid 132 (0.4) 1.0 99.0 Peritoneal fluid 101 (0.3)
28.6 71.4 Total: 32,966 (100.0) 20.0 80.0
TABLE-US-00004 TABLE 4 Example of microbial species for which tuf
and/or atpD and/or recA nucleic acids and/or sequences are used in
the present invention. Bacterial species Abiotrophia adiacens
Abiotrophia defectiva Achromobacter xylosoxidans subsp.
denitrificans Acetobacterium woodi Acetobacter aceti Acetobacter
altoacetigenes Acetobacter polyoxogenes Acholeplasma laidlawii
Acidothermus cellulolyticus Acidiphilum facilis Acinetobacter
baumannii Acinetobacter calcoaceticus Acinetobacter lwoffii
Actinomyces meyeri Aerococcus viridans Aeromonas hydrophila
Aeromonas salmonicida Agrobacterium radiobacter Agrobacterium
tumefaciens Alcaligenes faecalis subsp. faecalis Allochromatium
vinosum Anabaena variabilis Anacystis nidulans Anaerorhabdus
furcosus Aquifex aeolicus Aquifex pyrophilus Arcanobacterium
haemolyticum Archaeoglobus fulgidus Azotobacter vinelandii Bacillus
anthracis Bacillus cereus Bacillus firmus Bacillus halodurans
Bacillus megaterium Bacillus mycoides Bacillus pseudomycoides
Bacillus stearothermophilus Bacillus subtilis Bacillus
thuringiensis Bacillus weihenstephanensis Bacteroides distasonis
Bacteroides fragilis Bacteroides forsythus Bacteroides ovatus
Bacteroides vulgatus Bartonella henselae Bifidobacterium
adolescentis Bifidobacterium breve Bifidobacterium dentium
Bifidobacterium longum Blastochloris viridis Borrelia burgdorferi
Bordetella pertussis Bordetella bronchiseptica Brucella abortus
Brevibacterium linens Brevibacterium flavum Brevundimonas diminuta
Buchnera aphidicola Budvicia aquatica Burkholderia cepacia
Burkholderia mallei Burkholderia pseudomallei Buttiauxella agrestis
Butyrivibrio fibrisolvens Campylobacter coli Campylobacter curvus
Campylobacter fetus subsp. fetus Campylobacter fetus subsp.
venerealis Campylobacter gracilis Campylobacter jejuni
Campylobacter jejuni subsp. doylei Campylobacter jejuni subsp.
jejuni Campylobacter lari Campylobacter rectus Campylobacter
sputorum subsp. sputorum Campylobacter upsaliensis Cedecea davisae
Cedecea lapagei Cedecea neteri Chlamydia pneumoniae Chlamydia
psittaci Chlamydia trachomatis Chlorobium vibrioforme Chloroflexus
aurantiacus Chryseobacterium meningosepticum Citrobacter
amalonaticus Citrobacter braakii Citrobacter farmeri Citrobacter
freundii Citrobacter koseri Citrobacter sedlakii Citrobacter
werkmanii Citrobacter youngae Clostridium acetobutylicum
Clostridium beijerinckii Clostridium bifermentans Clostridium
botulinum Clostridium difficile Clostridium innocuum Clostridium
histolyticum Clostridium novyi Clostridium septicum Clostridium
perfringens Clostridium ramosum Clostridium tertium Clostridium
tetani Comamonas acidovorans Corynebacterium accolens
Corynebacterium bovis Corynebacterium cervicis Corynebacterium
diphtheriae Corynebacterium flavescens Corynebacterium genitalium
Corynebacterium glutamicum Corynebacterium jeikeium Corynebacterium
kutscheri Corynebacterium minutissimum Corynebacterium mycetoides
Corynebacterium pseudodiphtheriticum Corynebacterium
pseudogenitalium Corynebacterium pseudotuberculosis Corynebacterium
renale Corynebacterium striatum Corynebacterium ulcerans
Corynebacterium urealyticum Corynebacterium xerosis Coxiella
burnetii Cytophaga lytica Deinococcus radiodurans Deinonema sp.
Edwardsiella hoshinae Edwardsiella tarda Ehrlichia canis Ehrlichia
risticii Eikenella corrodens Enterobacter aerogenes Enterobacter
agglomerans Enterobacter amnigenus Enterobacter asburiae
Enterobacter cancerogenus Enterobacter cloacae Enterobacter
gergoviae Enterobacter hormaechei Enterobacter sakazakii
Enterococcus avium Enterococcus casseliflavus Enterococcus cecorum
Enterococcus columbae Enterococcus dispar Enterococcus durans
Enterococcus faecalis Enterococcus faecium Enterococcus flavescens
Enterococcus gallinarum Enterococcus hirae Enterococcus malodoratus
Enterococcus mundtii Enterococcus pseudoavium Enterococcus
raffinosus Enterococcus saccharolyticus Enterococcus solitarius
Enterococcus sulfureus Clostridium sordellii Erwinia amylovora
Erwinia carotovora Escherichia coli Escherichia fergusonii
Escherichia hermannii Escherichia vulneris Eubacterium lentum
Eubacterium nodatum Ewingella americana Francisella tularensis
Frankia alni Fervidobacterium islandicum Fibrobacter succinogenes
Flavobacterium ferrigeneum Flexistipes sinusarabici Fusobacterium
gonidiaformans Fusobacterium necrophorum subsp. necrophorum
Fusobacterium nucleatum subsp. polymorphum Gardnerella vaginalis
Gemella haemolysans Gemella morbillorum Globicatella sanguis
Gloeobacter violaceus Gloeothece sp. Gluconobacter oxydans
Haemophilus actinomycetemcomitans Haemophilus aphrophilus
Haemophilus ducreyi Haemophilus haemolyticus Haemophilus influenzae
Haemophilus parahaemolyticus Haemophilus parainfluenzae Haemophilus
paraphrophilus Haemophilus segnis Hafnia alvei Halobacterium
marismortui Halobacterium salinarum Haloferax volcanii Helicobacter
pylori Herpetoshiphon aurantiacus Kingella kingae Klebsiella
ornithinolytica Klebsiella oxytoca Klebsiella planticola Klebsiella
pneumoniae subsp. ozaenae Klebsiella pneumoniae subsp. pneumoniae
Klebsiella pneumoniae subsp. rhinoscleromatis Klebsiella terrigena
Kluyvera ascorbata Kluyvera cryocrescens Kluyvera georgiana Kocuria
kristinae Lactobacillus acidophilus Lactobacillus garvieae
Lactobacillus paracasei Lactobacillus casei subsp. casei
Lactococcus garvieae Lactococcus lactis Lactococcus lactis subsp.
lactis Legionella micdadei Legionella pneumophila subsp.
pneumophila Leminorella grimontii Leminorella richardii Leptospira
biflexa Leptospira interrogans Leuconostoc mesenteroides subsp.
dextranicum Listeria innocua Listeria ivanovii Listeria
monocytogenes Listeria seeligeri Macrococcus caseolyticus
Magnetospirillum magnetotacticum Megamonas hypermegale
Methanobacterium thermoautotrophicum Methanococcus jannaschii
Methanococcus vannielii Methanosarcina barkeri Methanosarcina
jannaschii Methylobacillus flagellatum
Methylomonas clara Micrococcus luteus Micrococcus lylae Mitsuokella
multacidus Mobiluncus curtisii subsp. holmesii Moellerella
thermoacetica Moellerella wisconsensis Moorella thermoacetica
Moraxella catarrhalis Moraxella osloensis Morganella morganii
subsp. morganii Mycobacterium avium Mycobacterium bovis
Mycobacterium gordonae Mycobacterium kansasii Mycobacterium leprae
Mycobacterium terrae Mycobacterium tuberculosis Mycoplasma
capricolum Mycoplasma gallisepticum Mycoplasma genitalium
Mycoplasma hominis Mycoplasma pirum Mycoplasma mycoides Mycoplasma
pneumoniae Mycoplasma pulmonis Mycoplasma salivarium Myxococcus
xanthus Neisseria animalis Neisseria canis Neisseria cinerea
Neisseria cuniculi Neisseria elongata subsp. elongata Neisseria
elongata subsp. intermedia Neisseria flava Neisseria flavescens
Neisseria gonorrhoeae Neisseria lactamica Leclercia adecarboxylata
Neisseria meningitidis Neisseria mucosa Neisseria perflava
Neisseria pharyngis var. flava Neisseria polysaccharea Neisseria
sicca Neisseria subflava Neisseria weaveri Obesumbacterium proteus
Ochrobactrum anthropi Pantoea agglomerans Pantoea dispersa
Paracoccus denitrificans Pasteurella multocida Pectinatus
frisingensis Peptococcus niger Peptostreptococcus anaerobius
Peptostreptococcus asaccharolyticus Peptostreptococcus prevotii
Phormidium ectocarpi Pirellula marina Planobispora rosea
Plesiomonas shigelloides Plectonema boryanum Porphyromonas
asaccharolytica Porphyromonas gingivalis Pragia fontium Prevotella
buccalis Prevotella melaninogenica Prevotella oralis Prevotella
ruminocola Prochlorothrix hollandica Propionibacterium acnes
Propionigenium modestum Proteus mirabilis Proteus penneri Proteus
vulgaris Providencia alcalifaciens Providencia rettgeri Providencia
rustigianii Providencia stuartii Pseudomonas aeruginosa Pseudomonas
fluorescens Pseudomonas putida Pseudomonas stutzeri Psychrobacter
phenylpyruvicum Pyrococcus abyssi Rahnella aquatilis Rickettsia
prowazekii Rhizobium leguminosarum Rhizobium phaseoli Rhodobacter
capsulatus Rhodobacter sphaeroides Rhodopseudomonas palustris
Rhodospirillum rubrum Ruminococcus albus Ruminococcus bromii
Salmonella bongori Salmonella choleraesuis subsp. arizonae
Salmonella choleraesuis subsp choleraesuis Salmonella choleraesuis
subsp. diarizonae Salmonella choleraesuis subsp. houtenae
Salmonella choleraesuis subsp. indica Salmonella choleraesuis
subsp. salamae Serpulina hyodysenteriae Serratia ficaria Serratia
fonticola Serratia grimesii Serratia liquefaciens Serratia
marcescens Serratia odorifera Serratia plymuthica Serratia rubidaea
Shewanella putrefaciens Shigella boydii Shigella dysenteriae
Shigella flexneri Shigella sonnei Sinorhizobium meliloti
Spirochaeta aurantia Staphylococcus aureus Staphylococcus aureus
subsp. aureus Staphylococcus auricularis Staphylococcus capitis
subsp. capitis Staphylococcus cohnii subsp. cohnii Staphylococcus
epidermidis Staphylococcus haemolyticus Staphylococcus hominis
Staphylococcus hominis subsp. hominis Staphylococcus lugdunensis
Staphylococcus saprophyticus Staphylococcus sciuri subsp. sciuri
Staphylococcus simulans Staphylococcus warneri Stigmatella
aurantiaca Stenotrophomonas maltophilia Streptococcus acidominimus
Streptococcus agalactiae Streptococcus anginosus Streptococcus
bovis Streptococcus cricetus Streptococcus cristatus Streptococcus
downei Streptococcus dysgalactiae Streptococcus equi subsp. equi
Streptococcus ferus Streptococcus gordonii Streptococcus macacae
Streptococcus mitis Streptococcus mutans Streptococcus oralis
Streptococcus parasanguinis Streptococcus pneumoniae Streptococcus
pyogenes Streptococcus ratti Streptococcus salivarius Streptococcus
salivarius subsp. thermophilus Streptococcus sanguinis
Streptococcus sobrinus Streptococcus suis Streptococcus uberis
Streptococcus vestibularis Streptomyces anbofaciens Streptomyces
aureofaciens Streptomyces cinnamoneus Streptomyces coelicolor
Streptomyces collinus Streptomyces lividans Streptomyces netropsis
Streptomyces ramocissimus Streptomyces rimosus Streptomyces
venezuelae Succinivibrio dextrinosolvens Synechococcus sp.
Synechocystis sp. Tatumella ptyseos Taxeobacter occealus
Tetragenococcus halophilus Thermoplasma acidophilum Thermotoga
maritima Thermus aquaticus Thermus thermophilus Thiobacillus
ferrooxidans Thiomonas cuprina Trabulsiella guamensis Treponema
pallidum Ureaplasma urealyticum Veillonella parvula Vibrio
alginolyticus Vibrio anguillarum Vibrio cholerae Vibrio mimicus
Wolinella succinogenes Xanthomonas citri Xanthomonas oryzae
Xenorhabdus bovieni Xenorhabdus nematophilus Yersinia bercovieri
Yersinia enterocolitica Yersinia frederiksensii Yersinia intermedia
Yersinia pestis Yersinia pseudotuberculosis Yersinia rohdei
Yokenella regensburgei Zoogloea ramigera
TABLE-US-00005 TABLE 4 Example of microbial species for which tuf
and/or atpD and/or recA nucleic acids and/or sequences are used in
the present invention (continued) Fungal species Absidia
corymbifera Absidia glauca Alternaria alternate Arxula
adeninivorans Aspergillus flavus Aspergillus fumigatus Aspergillus
nidulans Aspergillus niger Aspergillus oryzae Aspergillus terreus
Aspergillus versicolor Aureobasidium pullulans Basidiobolus ranarum
Bipolaris hawaiiensis Bilophila wadsworthia Blastoschizomyces
capitatus Blastomyces dermatitidis Candida albicans Candida
catenulata Candida dubliniensis Candida famata Candida glabrata
Candida guilliermondii Candida haemulonii Candida inconspicua
Candida kefyr Candida krusei Candida lambica Candida lusitaniae
Candida norvegica Candida norvegensis Candida parapsilosis Candida
rugosa Candida sphaerica Candida tropicalis Candida utilis Candida
viswanathii Candida zeylanoides Cladophialophora carrionii
Coccidioides immitis Coprinus cinereus Cryptococcus albidus
Cryptococcus humicolus Cryptococcus laurentii Cryptococcus
neoformans Cunninghamella bertholletiae Curvularia lunata
Emericella nidulans Emmonsia parva Eremothecium gossypii Exophiala
dermatitidis Exophiala jeanselmei Exophiala moniliae Exserohilum
rostratum Eremothecium gossypii Fonsecaea pedrosoi Fusarium
moniliforme Fusarium oxysporum Fusarium solani Geotrichum sp.
Histoplasma capsulatum Hortaea werneckii Issatchenkia orientalis
Kudrjanzev Kluyveromyces lactis Malassezia furfur Malassezia
pachydermatis Malbranchea filamentosa Metschnikowia pulcherrima
Microsporum audouinii Microsporum canis Mucor circinelloides
Neurospora crassa Paecilomyces lilacinus Paracoccidioides
brasiliensis Penicillium marneffei Phialaphora verrucosa Pichia
anomala Piedraia hortai Podospora anserina Podospora curvicolla
Puccinia graminis Pseudallescheria boydii Reclinomonas americana
Rhizomucor racemosus Rhizopus oryzae Rhodotorula minuta Rhodotorula
mucilaginosa Saccharomyces cerevisiae Saksenaea vasiformis
Schizosaccharomyces pombe Scopulariopsis koningii Sordaria
macrospora Sporobolomyces salmonicolor Sporothrix schenckii
Stephanoascus ciferrii Syncephalastrum racemosum Trichoderma reesei
Trichophyton mentagrophytes Trichophyton rubrum Trichophyton
tonsurans Trichosporon cutaneum Ustilago maydis Wangiella
dermatitidis Yarrowia lipolytica Parasitical species Babesia
bigemina Babesia bovis Babesia microti Blastocystis hominis
Crithidia fasciculata Cryptosporidium parvum Entamoeba histolytica
Giardia lamblia Kentrophoros sp. Leishmania aethiopica Leishmania
amazonensis Leishmania braziliensis Leishmania donovani Leishmania
infantum Leishmania enriettii Leishmania gerbilli Leishmania
guyanensis Leishmania hertigi Leishmania major Leishmania mexicana
Leishmania panamensis Leishmania tarentolae Leishmania tropica
Neospora caninum Onchocerca volvulus Plasmodium berghei Plasmodium
falciparum Plasmodium knowlesi Porphyra purpurea Toxoplasma gondii
Treponema pallidum Trichomonas tenax Trichomonas vaginalis
Trypanosoma brucei Trypanosoma brucei subsp. brucei Trypanosoma
congolense Trypanosoma cruzi
TABLE-US-00006 TABLE 5 Antimicrobial agents resistance genes
selected for diagnostic purposes SEQ Gene Antimicrobial agent
Bacteria.sup.1 ACCESSION NO. ID NO. aac(3)-Ib.sup.2 Aminoglycosides
Enterobacteriaceae L06157 Pseudomonads aac(3)-IIb.sup.2
Aminoglycosides Enterobacteriaceae, M97172 Pseudomonads
aac(3)-IVa.sup.2 Aminoglycosides Enterobacteriaceae X01385
aac(3)-VIa.sup.2 Aminoglycosides Enterobacteriaceae, M88012
Pseudomonads aac(2')-1a.sup.2 Aminoglycosides Enterobacteriaceae,
X04555 Pseudomonads aac(6')-aph(2'').sup.2 Aminoglycosides
Enterococcus sp., 83-86.sup.3 Staphylococcus sp. aac(6')-Ia,.sup.2
Aminoglycosides Enterobacteriaceae, M18967 Pseudomonads
aac(6')-Ic.sup.2 Aminoglycosides Enterobacteriaceae, M94066
Pseudomonads aac(6')-IIa.sup.2 Aminoglycosides Pseudomonads
112.sup.4 aadB [ant(2'')-Ia.sup.2] Aminoglycosides
Enterobacteriaceae 53-54.sup.3 aacC1 [aac(3)-Ia.sup.2]
Aminoglycosides Pseudomonads 55-56.sup.3 aacC2 [aac(3)-IIa.sup.2]
Aminoglycosides Pseudomonads 57-58.sup.3 aacC3 [aac(3)-III.sup.2]
Aminoglycosides Pseudomonads 59-60.sup.3 aacA4 [aac(6')-Ib.sup.2]
Aminoglycosides Pseudomonads 65-66.sup.3 ant(3'')-Ia.sup.2
Aminoglycosides Enterobacteriaceae, X02340 Enterococcus sp., M10241
Staphylococcus sp. ant(4')-Ia.sup.2 Aminoglycosides Staphylococcus
sp. V01282 aph(3')-Ia.sup.2 Aminoglycosides Enterobacteriaceae,
J01839 Pseudomonads aph(3')-IIa.sup.2 Aminoglycosides
Enterobacteriaceae, V00618 Pseudomonads aph(3')-IIIa.sup.2
Aminoglycosides Enterococcus sp., V01547 Staphylococcus sp.
aph(3')-VIa.sup.2 Aminoglycosides Enterobacteriaceae, X07753
Pseudomonads rpsL.sup.2 Streptomycin M. tuberculosis, X80120 M.
avium complex U14749 X70995 L08011 bla.sub.OXA.sup.5,6
.beta.-lactams Enterobacteriaceae, Y10693 110.sup.4 Pseudomonads
AJ238349 AJ009819 X06046 X03037 X07260 U13880 X75562 AF034958
J03427 Z22590 U59183 L38523 U63835 AF043100 AF060206 U85514
AF043381 AF024602 AF064820 bla.sub.ROB.sup.5 .beta.-lactams
Haemophilus sp. 45-48.sup.3 bla.sub.SHV.sup.5,6 .beta.-lactams
Enterobacteriacea, AF124984 41-44.sup.3 Pseudomonas aeruginosa
AF148850 M59181 X98099 M33655 AF148851 X53433 L47119 AF074954
X53817 AF096930 X55640 Y11069 U20270 U92041 S82452 X98101 X98105
AF164577 AJ011428 AF116855 AB023477 AF293345 AF227204 AF208796
AF132290 bla.sub.TEM.sup.5,6 .beta.-lactams Enterobacteriaceae,
AF012911 37-40.sup.3 Neisseria sp., U48775 Haemophilus sp. AF093512
AF052748 X64523 Y13612 X57972 AF157413 U31280 U36911 U48775 V00613
X97254 AJ012256 X04515 AF126482 U09188 M88143 Y14574 AF188200
AJ251946 Y17581 Y17582 Y17583 M88143 U37195 Y17584 X64523 U95363
Y10279 Y10280 Y10281 AF027199 AF104441 AF104442 AF062386 X57972
AF047171 AF188199 AF157553 AF190694 AF190695 AF190693 AF190692
bla.sub.SHV.sup.5,6 .beta.-lactams Enterobacteriacea, AF124984
41-44.sup.3 Pseudomonas aeruginosa AF148850 M59181 X98099 M33655
AF148851 X53433 L47119 AF074954 X53817 AF096930 X55640 Y11069
U20270 U92041 S82452 X98101 X98105 AF164577 AJ011428 AF116855
AB023477 AF293345 AF227204 AF208796 AF132290 bla.sub.TEM.sup.5,6
.beta.-lactams Enterobacteriaceae, AF012911 37-40.sup.3 Neisseria
sp., U48775 Haemophilus sp. AF093512 AF052748 X64523 Y13612 X57972
AF157413 U31280 U36911 U48775 V00613 X97254 AJ012256 X04515
AF126482 U09188 M88143 Y14574 AF188200 AJ251946 Y17581 Y17582
Y17583 M88143 U37195 Y17584 X64523 U95363 Y10279 Y10280 Y10281
AF027199 AF104441 AF104442 AF062386 X57972 AF047171 AF188199
AF157553 AF190694 AF190695 AF190693 AF190692 bla.sub.CARB.sup.5
.beta.-lactams Pseudomonas sp., J05162 Enterobacteriaceae S46063
M69058 U14749 D86225 D13210 Z18955 AF071555 AF153200 AF030945
bla.sub.CTX-M-1.sup.5 .beta.-lactams Enterobacteriaceae X92506
bla.sub.CTX-M-2.sup.5 .beta.-lactams Enterobacteriaceae X92507
bla.sub.CMY-2.sup.7 .beta.-lactams Enterobacteriaceae X91840
AJ007826 AJ011293 AJ011291 Y17716 Y16783 Y16781 Y15130 U77414
S83226 Y15412 X78117 bla.sub.IMP.sup.5 .beta.-lactams
Enterobacteriaceae, AJ223604 Pseudomonas aeruginosa S71932 D50438
D29636 X98393 AB010417 D78375 bla.sub.PER-1.sup.5 .beta.-lactams
Enterobacteriaceae, Z21957 Pseudomodanaceae bla.sub.PER-2.sup.7
.beta.-lactams Enterobacteriaceae X93314 blaZ.sup.12 .beta.-lactams
Enterococcus sp., 111.sup.4 Staphylococcus sp. mecA.sup.12
.beta.-lactams Staphylococcus sp. 97-98.sup.3 pbp1a.sup.13
.beta.-lactams Streptococcus pneumoniae M90527 1004-1018, X67872
1648, 2056-2064, AB006868 2273-2276 AB006874 X67873
AB006878 AB006875 AB006877 AB006879 AF046237 AF046235 AF026431
AF046232 AF046233 AF046236 X67871 Z49095 AF046234 AB006873 X67866
X67868 AB006870 AB006869 AB006872 X67870 AB006871 X67867 X67869
AB006876 AF046230 AF046238 Z49094 pbp2b.sup.13 .beta.-lactams
Streptococcus pneumoniae X16022 1019-1033 M25516 M25518 M25515
U20071 U20084 U20082 U20067 U20079 Z22185 U20072 pbp2b.sup.13
.beta.-lactams Streptococcus pneumoniae U20083 U20081 M25522 U20075
U20070 U20077 U20068 Z22184 U20069 U20078 M25521 M25525 M25519
Z21981 M25523 M25526 U20076 U20074 M25520 M25517 M25524 Z22230
U20073 U20080 pbp2x.sup.13 .beta.-lactams Streptococcus pneumoniae
X16367 1034-1048 X65135 AB011204 AB011209 AB011199 AB011200
AB011201 AB011202 AB011198 AB011208 AB011205 AB015852 AB011210
AB015849 AB015850 AB015851 AB015847 AB015846 AB011207 AB015848
Z49096 int .beta.-lactams, Enterobacteriaceae, 99-102.sup.3
trimethoprim sul aminoglycosides, Pseudomonads 103-106.sup.3
antiseptic, chloramphenicol ermA.sup.14 Macrolides, Staphylococcus
sp. 113.sup.4 lincosamides, streptogramin B ermB.sup.14 Macrolides,
Enterobacteriaceae, 114.sup.4 lincosamides, Staphylococcus sp.
streptogramin B Enterococcus sp. Streptococcus sp. ermC.sup.14
Macrolides, Enterobacteriaceae, 115.sup.4 lincosamides,
Staphylococcus sp. streptogramin B ereA.sup.12 Macrolides
Enterobacteriaceae, M11277 Staphylococcus sp. E01199 AF099140
ereB.sup.12 Macrolides Enterobacteriaceae A15097 Staphylococcus sp.
X03988 msrA.sup.12 Macrolides Staphylococcus sp. 77-80.sup.3 mefA,
mefE.sup.8 Macrolides Streptococcus sp. U70055 U83667 mphA.sup.8
Macrolides Enterobacteriaceae, D16251 Staphylococcus sp. U34344
U36578 linA/linA'.sup.9 Lincosamides Staphylococcus sp. J03947
M14039 A15070 E01245 linB.sup.10 Lincosamides Enterococcus faecium
AF110130 AJ238249 vga.sup.15 Streptrogramin Staphylococcus sp.
M90056 89-90.sup.3 U82085 vgb.sup.15 Streptrogramin Staphylococcus
sp. M36022 M20219 AF015628 vat.sup.15 Streptrogramin Staphylococcus
sp. L07778 87-88.sup.3 vatB.sup.15 Streptrogramin Staphylococcus
sp. U19459 L38809 satA.sup.15 Streptrogramin Enterococcus faecium
L12033 81-82.sup.3 mupA.sup.12 Mupirocin Staphylococcus aureus
X75439 X59478 X59477 gyrA.sup.16 Quinolones Gram-positive and
X95718 1255, 1607-1608, gram-negative bacteria X06744 1764-1776,
X57174 2013-2014, X16817 2277-2280 X71437 AF065152 AF060881 D32252
parC/grlA.sup.16 Quinolones Gram-positive and AB005036 1777-1785
gram-negative bacteria AF056287 X95717 AF129764 AB017811 AF065152
parE/grlB.sup.16 Quinolones Gram-positive bacteria X95717 AF065153
AF058920 norA.sup.16 Quinolones Staphylococcus sp. D90119 M80252
M97169 mexR (nalB).sup.16 Quinolones Pseudomonas aeruginosa U23763
nfxB.sup.16 Quinolones Pseudomonas aeruginosa X65646 cat.sup.12
Chloramphenicol Gram-positive and M55620 gram-negative bacteria
X15100 A24651 M28717 A00568 A00569 X74948 Y00723 A24362 A00569
M93113 M62822 M58516 V01277 X02166 M77169 X53796 J01841 X07848
ppflo-like Chloramphenicol AF071555 embB.sup.17 Ethambutol
Mycobacterium tuberculosis U68480 pncA.sup.17 Pyrazinamide
Mycobacterium tuberculosis U59967 rpoB.sup.17 Rifampin
Mycobacterium tuberculosis AF055891 AF055892 S71246 L27989 AF055893
inhA.sup.17 Isoniazid Mycobacterium tuberculosis AF106077 U02492
vanA.sup.12 Vancomycin Enterococcus sp. 67-70.sup.3 1049-1057
vanB.sup.12 Vancomycin Enterococcus sp. 116.sup.4 vanC1.sup.12
Vancomycin Enterococcus gallinarum 117.sup.4 1058-1059 vanC2.sup.12
Vancomycin Enterococcus casseliflavus U94521 1060-1063 U94522
U94523 U94524 U94525 L29638 vanC3.sup.12 Vancomycin Enterococcus
flavescens L29639 1064-1066 U72706 vanD.sup.18 Vancomycin
Enterococcus faecium AF130997 vanE.sup.12 Vancomycin Enterococcus
faecium AF136925 tetB.sup.19 Tetracycline Gram-negative bacteria
J01830 AF162223 AP000342 S83213 U81141 V00611 tetM.sup.19
Tetracycline Gram-negative and X52632 Gram-positive bacteria
AF116348 U50983 X92947 M211136 U08812 X04388 sul II.sup.20
Sulfonamides Gram-negative bacteria M36657 AF017389 AF017391
dhfrIa.sup.20 Trimethoprim Gram-negative bacteria AJ238350 X17477
K00052 U09476 X00926 dhfrIb.sup.20 Trimethoprim Gram-negative
bacteria Z50805 Z50804 dhfrV.sup.20 Trimethoprim Gram-negative
bacteria X12868 dhfrVI.sup.20 Trimethoprim Gram-negative bacteria
Z86002 dhfrVII.sup.20 Trimethoprim Gram-negative bacteria U31119
AF139109 X58425 dhfrVIII.sup.20 Trimethoprim Gram-negative bacteria
U10186 U09273 dhfrIX.sup.20 Trimethoprim Gram-negative bacteria
X57730 dhfrXII.sup.20 Trimethoprim Gram-negative bacteria Z21672
AF175203 AF180731 M84522 dhfrXIII.sup.20 Trimethoprim Gram-negative
bacteria Z50802 dhfrXV.sup.20 Trimethoprim Gram-negative bacteria
Z83331 dhfrXVII.sup.20 Trimethoprim Gram-negative bacteria AF170088
AF180469 AF169041 dfrA.sup.20 Trimethoprim Staphylococcus sp.
AF045472 U40259 AF051916 X13290 Y07536 Z16422 Z48233 .sup.1Bacteria
having high incidence for the specified antibiotic resistance gene.
The presence of the antibiotic resistance genes in other bacteria
is not excluded. .sup.2Shaw, K. J., P. N. Rather, R. S. Hare, and
G. H. Miller. 1993. Molecular genetics of aminoglycoside resistance
genes and familial relationships of the aminoglycoside-modifying
enzymes. Microbial. Rev. 57: 138-163. .sup.3Antibiotic resistance
genes from our assigned U.S. Pat. No. 6,001,564 for which we have
selected PCR primer pairs. .sup.4These SEQ ID NOs. refer to a
previous patent (publication
WO98/20157). .sup.5Bush, K., G. A. Jacoby and A. Medeiros. 1995. A
functional classification scheme for .beta.-lactamase and its
correlation with molecular structure. Antimicrob. Agents.
Chemother. 39: 1211-1233. .sup.6Nucleotide mutations in
bla.sub.SHV, bla.sub.TEM, and bla.sub.OXA, are associated with
extended-spectrum .beta.-lactamase or inhibitor-resistant
.beta.-lactamase. .sup.7Bauerfeind, A., Y. Chong, and K. Lee. 1998.
Plasmid-encoded AmpC beta-lactamases: how far have we gone 10 ears
after discovery? Yonsei Med. J. 39: 520-525. .sup.8Sutcliffe. J.,
T. Grebe, A. Tait-Kamradt, and L. Wondrack. 1996. Detection of
erythromycin-resistant determinants by PCR. Antimicrob. Agent
Chemother. 40: 2562-2566. .sup.9Leclerc, R., A., Brisson-Noel, J.
Duval, and P. Courvalin. 1991. Phenotypic expression and genetic
heterogeneity of lincosamide inactivation in Staphylococcus sp.
Antimicrob. Agents. Chemother. 31: 1887-1891. .sup.10Bozdogan, B.,
L. Berrezouga, M.-S. Kuo, D. A. Yurek, K. A. Farley, B. J.
Stockman, and R. Leclercq. 1999. A new gene, linB, conferring
resistance to lincosamides by nucleotidylation in Enterococcus
faecium HM1025. Antimicrob. Agents. Chemother. 43: 925-929.
.sup.11Cockerill III, F. R. 1999. Genetic methods for assessing
antimicrobial resistance. Antimicrob. Agents. Chemother. 43:
199-212. .sup.12Tenover, F. C., T. Popovic, and O Olsvik. 1996.
Genetic methods for detecting antibacterial resistance genes. pp.
1368-1378. In Murray, P. R., E. J. Baron, M. A. Pfaller, F. C.
Tenover, R. H. Yolken (eds). Manual of clinical microbiology. 6th
ed., ASM Press, Washington, D. C. USA .sup.13Dowson, C. G., T. J.
Tracey, and B. G. Spratt. 1994. Origin and molecular epidemiology
of penicillin-binding-protein-mediated resistance to .beta.-lactam
antibiotics. Trends Molec. Microbiol. 2: 361-366. .sup.14Jensen, L.
B., N. Frimodt-Moller, F. M. Aarestrup. 1999. Presence of erm gene
classes in Gram-positive bacteria of animal and human origin in
Denmark. FEMS Microbiol. 170: 151-158: .sup.15Thal, L. A., and M.
J. Zervos. 1999. Occurrence and epidemiology of resistance to
virginimycin and streptrogramins. J. Antimicrob. Chemother. 43:
171-176. .sup.16Martinez J. L., A. Alonso, J. M. Gomez-Gomez, and
F. Baquero. 1998. Quinolone resistance by mutations in chromosomal
gyrase genes. Just the tip of the iceberg? J. Antimicrob.
Chemother. 42: 683-688 .sup.17Cockerill III, F. R. 1999. Genetic
methods for assessing antimicrobial resistance. Antimicrob. Agents.
Chemother. 43: 199-212. .sup.18Casadewall, B. and P. Courvalin.
1999 Characterization of the vanD glycopeptide resistance gene
cluster from Enterococcus faecium BM 4339. J. Bacteriol. 181:
3644-3648. .sup.19Roberts, M. C. 1999. Genetic mobility and
distribution of tetracycline resistance determinants. Ciba Found.
Symp. 207: 206-222. .sup.20Huovinen, P., L. Sundstrom, G. Swedberg,
and O. Skold. 1995. Trimethoprim and sulfonamide
resistance.Antimicrob. Agent Chemother. 39: 279-289.
TABLE-US-00007 TABLE 6 List of bacterial toxins selected for
diagnostic purposes. Organism Toxin Accession number Actinobacillus
actinomycetemcomitans Cytolethal distending toxin (cdtA, cdtB,
cdtC) AF006830 Leukotoxin (ltxA) M27399 Actinomyces pyogenes
Hemolysin (pyolysin) U84782 Aeromonas hydrophila Aerolysin (aerA)
M16495 Haemolysin (hlyA) U81555 Cytotonic enterotoxin (alt) L77573
Bacillus anthracis Anthrax toxin (cya) M23179 Bacillus cereus
Enterotoxin (bceT) D17312 AF192766, AF192767 Enterotoxic hemolysin
BL AJ237785 Non-haemolytic enterotoxins A, B and C (nhe) Y19005
Bacillus mycoides Hemolytic enterotoxin HBL AJ243150 to AJ243153
Bacillus pseudomycoides Hemolytic enterotoxin HBL AJ243154 to
AJ243156 Bacteroides fragilis Enterotoxin (bftP) U67735 Matrix
metalloprotease/enterotoxin (fragilysin) S75941, AF038459
Metalloprotease toxin-2 U90931 AF081785 Metalloprotease toxin-3
AF056297 Bordetella bronchiseptica Adenylate cyclase hemolysin
(cyaA) Z37112, U22953 Dermonecrotic toxin (dnt) U59687 AB020025
Bordetella pertussis Pertussis toxin (S1 subunit, tox) AJ006151
AJ006153 AJ006155 AJ006157 AJ006159 AJ007363 M14378, M16494
AJ007364 M13223 X16347 Adenyl cyclase (cya) 18323 Dermonecrotic
toxin (dnt) U10527 Campylobacter jejuni Cytolethal distending toxin
(cdtA, cdtB, cdtC) U51121 Citrobacter freundii Shiga-like toxin
(slt-llcA) X67514, S53206 Clostridium botulinum Botulism toxin
(BoNT) (A, B, E and F serotypes X52066, X52088 are neurotoxic for
humans; the other serotypes X73423 have not been considered) M30196
X70814 X70819 X71343 Z11934 X70817 M81186 X70818 X70815 X62089
X62683 S76749 X81714 X70816 X70820 X70281 L35496 M92906 Clostridium
difficile A toxin (enterotoxin) (tcdA) (cdtA) AB012304 AF053400
Y12616 X51797 X17194 M30307 B toxin (cytotoxin) (toxB) (cdtB)
Z23277 X53138 Clostridium perfringens Alpha (phospholipase C) (cpa)
L43545 L43546 L43547 L43548 X13608 X17300 D10248 Beta
(dermonecrotic protein) (cpb) L13198 X83275 L77965 Enterotoxin
(cpe) AJ000766 M98037 X81849 X71844 Y16009 Enterotoxin pseudogene
(not expressed) AF037328 AF037329 AF037330 Epsilon toxin (etxD)
M80837 M95206 X60694 Iota (Ia and Ib) X73562 Lambda
(metalloprotease) D45904 Theta (perfringolysin O) M36704
Clostridium sordellii Cytotoxin L X82638 Clostridium tetani Tetanos
toxin X06214 X04436 Corynebacterium diphtheriae Diphtheriae toxin
X00703 Corynebacterium pseudotuberculosis Phospholipase C A21336
Eikenella corrodens lysine decarboxylase (cadA) U89166 Enterobacter
cloacae Shiga-like toxin II Z50754, U33502 Enterococcus faecalis
Cytolysin B (cylB) M38052 Escherichia coli (EHEC) Hemolysin toxin
(hlyA and ehxA) AF043471 X94129 X79839 X86087 AB011549 AF074613
TABLE-US-00008 TABLE 7 Origin of the nucleic acids and/or sequences
in the sequence listing. SEQ ID NO. Archaeal, bacterial, fungal or
parasitical species Source Gene* 1 Acinetobacter baumannii This
patent tuf 2 Actinomyces meyeri This patent tuf 3 Aerococcus
viridans This patent tuf 4 Achromobacter xylosoxidans subsp.
denitrificans This patent tuf 5 Anaerorhabdus furcosus This patent
tuf 6 Bacillus anthracis This patent tuf 7 Bacillus cereus This
patent tuf 8 Bacteroides distasonis This patent tuf 9 Enterococcus
casseliflavus This patent tuf 10 Staphylococcus saprophyticus This
patent tuf 11 Bacteroides ovatus This patent tuf 12 Bartonella
henselae This patent tuf 13 Bifidobacterium adolescentis This
patent tuf 14 Bifidobacterium dentium This patent tuf 15 Brucella
abortus This patent tuf 16 Burkholderia cepacia This patent tuf 17
Cedecea davisae This patent tuf 18 Cedecea neteri This patent tuf
19 Cedecea lapagei This patent tuf 20 Chlamydia pneumoniae This
patent tuf 21 Chlamydia psittaci This patent tuf 22 Chlamydia
trachomatis This patent tuf 23 Chryseobacterium meningosepticum
This patent tuf 24 Citrobacter amalonaticus This patent tuf 25
Citrobacter braakii This patent tuf 26 Citrobacter koseri This
patent tuf 27 Citrobacter farmeri This patent tuf 28 Citrobacter
freundii This patent tuf 29 Citrobacter sedlakii This patent tuf 30
Citrobacter werkmanii This patent tuf 31 Citrobacter youngae This
patent tuf 32 Clostridium perfringens This patent tuf 33 Comamonas
acidovorans This patent tuf 34 Corynebacterium bovis This patent
tuf 35 Corynebacterium cervicis This patent tuf 36 Corynebacterium
flavescens This patent tuf 37 Corynebacterium kutscheri This patent
tuf 38 Corynebacterium minutissimum This patent tuf 39
Corynebacterium mycetoides This patent tuf 40 Corynebacterium
pseudogenitalium This patent tuf 41 Corynebacterium renale This
patent tuf 42 Corynebacterium ulcerans This patent tuf 43
Corynebacterium urealyticum This patent tuf 44 Corynebacterium
xerosis This patent tuf 45 Coxiella burnetii This patent tuf 46
Edwardsiella hoshinae This patent tuf 47 Edwardsiella tarda This
patent tuf 48 Eikenella corrodens This patent tuf 49 Enterobacter
aerogenes This patent tuf 50 Enterobacter agglomerans This patent
tuf 51 Enterobacter amnigenus This patent tuf 52 Enterobacter
asburiae This patent tuf 53 Enterobacter cancerogenus This patent
tuf 54 Enterobacter cloacae This patent tuf 55 Enterobacter
gergoviae This patent tuf 56 Enterobacter hormaechei This patent
tuf 57 Enterobacter sakazakii This patent tuf 58 Enterococcus
casseliflavus This patent tuf 59 Enterococcus cecorum This patent
tuf 60 Enterococcus dispar This patent tuf 61 Enterococcus durans
This patent tuf 62 Enterococcus faecalis This patent tuf 63
Enterococcus faecalis This patent tuf 64 Enterococcus faecium This
patent tuf 65 Enterococcus flavescens This patent tuf 66
Enterococcus gallinarum This patent tuf 67 Enterococcus hirae This
patent tuf 68 Enterococcus mundtii This patent tuf 69 Enterococcus
pseudoavium This patent tuf 70 Enterococcus raffinosus This patent
tuf 71 Enterococcus saccharolyticus This patent tuf 72 Enterococcus
solitarius This patent tuf 73 Enterococcus casseliflavus This
patent tuf (C) 74 Staphylococcus saprophyticus This patent unknown
75 Enterococcus flavescens This patent tuf (C) 76 Enterococcus
gallinarum This patent tuf (C) 77 Ehrlichia canis This patent tuf
78 Escherichia coli This patent tuf 79 Escherichia fergusonii This
patent tuf 80 Escherichia hermannii This patent tuf 81 Escherichia
vulneris This patent tuf 82 Eubacterium lentum This patent tuf 83
Eubacterium nodatum This patent tuf 84 Ewingella americana This
patent tuf 85 Francisella tularensis This patent tuf 86
Fusobacterium nucleatum subsp. polymorphum This patent tuf 87
Gemella haemolysans This patent tuf 88 Gemella morbillorum This
patent tuf 89 Haemophilus actinomycetemcomitans This patent tuf 90
Haemophilus aphrophilus This patent tuf 91 Haemophilus ducreyi This
patent tuf 92 Haemophilus haemolyticus This patent tuf 93
Haemophilus parahaemolyticus This patent tuf 94 Haemophilus
parainfluenzae This patent tuf 95 Haemophilus paraphrophilus This
patent tuf 96 Haemophilus segnis This patent tuf 97 Hafnia alvei
This patent tuf 98 Kingella kingae This patent tuf 99 Klebsiella
omithinolytica This patent tuf 100 Klebsiella oxytoca This patent
tuf 101 Klebsiella planticola This patent tuf 102 Klebsiella
pneumoniae subsp. ozaenae This patent tuf 103 Klebsiella pneumoniae
pneumoniae This patent tuf 104 Klebsiella pneumoniae subsp.
rhinoscleromatis This patent tuf 105 Kluyvera ascorbata This patent
tuf 106 Kluyvera cryocrescens This patent tuf 107 Kluyvera
georgiana This patent tuf 108 Lactobacillus casei subsp. casei This
patent tuf 109 Lactococcus lactis subsp. lactis This patent tuf 110
Leclercia adecarboxylata This patent tuf 111 Legionella micdadei
This patent tuf 112 Legionella pneumophila subsp. pneumophila This
patent tuf 113 Leminorella grimontii This patent tuf 114
Leminorella richardii This patent tuf 115 Leptospira interrogans
This patent tuf 116 Megamonas hypermegale This patent tuf 117
Mitsuokella multacidus This patent tuf 118 Mobiluncus curtisii
subsp. holmesii This patent tuf 119 Moellerella wisconsensis This
patent tuf 120 Moraxella catarrhalis This patent tuf 121 Morganella
morganii subsp. morganii This patent tuf 122 Mycobacterium
tuberculosis This patent tuf 123 Neisseria cinerea This patent tuf
124 Neisseria elongata subsp. elongata This patent tuf 125
Neisseria flavescens This patent tuf 126 Neisseria gonorrhoeae This
patent tuf 127 Neisseria lactamica This patent tuf 128 Neisseria
meningitidis This patent tuf 129 Neisseria mucosa This patent tuf
130 Neisseria sicca This patent tuf 131 Neisseria subflava This
patent tuf 132 Neisseria weaveri This patent tuf 133 Ochrobactrum
anthropi This patent tuf 134 Pantoea agglomerans This patent tuf
135 Pantoea dispersa This patent tuf 136 Pasteurella multocida This
patent tuf 137 Peptostreptococcus anaerobius This patent tuf 138
Peptostreptococcus asaccharolyticus This patent tuf 139
Peptostreptococcus prevotii This patent tuf 140 Porphyromonas
asaccharolytica This patent tuf 141 Porphyromonas gingivalis This
patent tuf 142 Pragia fontium This patent tuf 143 Prevotella
melaninogenica This patent tuf 144 Prevotella oralis This patent
tuf 145 Propionibacterium acnes This patent tuf 146 Proteus
mirabilis This patent tuf 147 Proteus penneri This patent tuf 148
Proteus vulgaris This patent tuf 149 Providencia alcalifaciens This
patent tuf 150 Providencia rettgeri This patent tuf 151 Providencia
rustigianii This patent tuf 152 Providencia stuartii This patent
tuf 153 Pseudomonas aeruginosa This patent tuf 154 Pseudomonas
fluorescens This patent tuf 155 Pseudomonas stutzeri This patent
tuf 156 Psychrobacter phenylpyruvicum This patent tuf 157 Rahnella
aquatilis This patent tuf 158 Salmonella choleraesuis subsp.
arizonae This patent tuf 159 Salmonella choleraesuis subsp.
choleraesuis This patent tuf serotype Choleraesuis 160 Salmonella
choleraesuis subsp. diarizonae This patent tuf 161 Salmonella
choleraesuis subsp. choleraesuis This patent tuf serotype
Heidelberg 162 Salmonella choleraesuis subsp. houtenae This patent
tuf 163 Salmonella choleraesuis subsp. indica This patent tuf 164
Salmonella choleraesuis subsp. salamae This patent tuf 165
Salmonella choleraesuis subsp. choleraesuis serotype Typhi This
patent tuf 166 Serratia fonticola This patent tuf 167 Serratia
liquefaciens This patent tuf 168 Serratia marcescens This patent
tuf 169 Serratia odorifera This patent tuf 170 Serratia plymuthica
This patent tuf 171 Serratia rubidaea This patent tuf 172 Shigella
boydii This patent tuf 173 Shigella dysenteriae This patent tuf 174
Shigella flexneri This patent tuf 175 Shigella sonnei This patent
tuf 176 Staphylococcus aureus This patent tuf 177 Staphylococcus
aureus This patent tuf 178 Staphylococcus aureus This patent tuf
179 Staphylococcus aureus This patent tuf 180 Staphylococcus aureus
subsp. aureus This patent tuf 181 Staphylococcus auricularis This
patent tuf 182 Staphylococcus capitis subsp. capitis This patent
tuf 183 Macrococcus caseolyticus This patent tuf 184 Staphylococcus
cohnii subsp. cohnii This patent tuf 185 Staphylococcus epidermidis
This patent tuf 186 Staphylococcus haemolyticus This patent tuf 187
Staphylococcus warneri This patent tuf 188 Staphylococcus
haemolyticus This patent tuf 189 Staphylococcus haemolyticus This
patent tuf 190 Staphylococcus haemolyticus This patent tuf 191
Staphylococcus hominis subsp. hominis This patent tuf 192
Staphylococcus warneri This patent tuf 193 Staphylococcus hominis
This patent tuf 194 Staphylococcus hominis This patent tuf 195
Staphylococcus hominis This patent tuf 196 Staphylococcus hominis
This patent tuf 197 Staphylococcus lugdunensis This patent tuf 198
Staphylococcus saprophyticus This patent tuf 199 Staphylococcus
saprophyticus This patent tuf 200 Staphylococcus saprophyticus This
patent tuf 201 Staphylococcus sciuri subsp. sciuri This patent tuf
202 Staphylococcus warneri This patent tuf 203 Staphylococcus
warneri This patent tuf 204 Bifidobacterium longum This patent tuf
205 Stenotrophomonas maltophilia This patent tuf 206 Streptococcus
acidominimus This patent tuf 207 Streptococcus agalactiae This
patent tuf 208 Streptococcus agalactiae This patent tuf 209
Streptococcus agalactiae This patent tuf 210 Streptococcus
agalactiae This patent tuf 211 Streptococcus anginosus This patent
tuf 212 Streptococcus bovis This patent tuf 213 Streptococcus
anginosus This patent tuf 214 Streptococcus cricetus This patent
tuf 215 Streptococcus cristatus This patent tuf 216 Streptococcus
downei This patent tuf 217 Streptococcus dysgalactiae This patent
tuf 218 Streptococcus equi subsp. equi This patent tuf 219
Streptococcus ferus This patent tuf 220 Streptococcus gordonii This
patent tuf 221 Streptococcus anginosus This patent tuf 222
Streptococcus macacae This patent tuf 223 Streptococcus gordonii
This patent tuf 224 Streptococcus mutans This patent tuf 225
Streptococcus parasanguinis This patent tuf 226 Streptococcus ratti
This patent tuf 227 Streptococcus sanguinis This patent tuf 228
Streptococcus sobrinus This patent tuf 229 Streptococcus suis This
patent tuf 230 Streptococcus uberis This patent tuf 231
Streptococcus vestibularis This patent tuf 232 Tatumella ptyseos
This patent tuf 233 Trabulsiella guamensis This patent tuf 234
Veillonella parvula This patent tuf 235 Yersinia enterocolitica
This patent tuf 236 Yersinia frederiksenii This patent tuf 237
Yersinia intermedia This patent tuf 238 Yersinia pestis This patent
tuf 239 Yersinia pseudotuberculosis This patent tuf 240 Yersinia
rohdei This patent tuf 241 Yokenella regensburgei This patent
tuf
242 Achromobacter xylosoxidans subsp. denitrificans This patent
atpD 243 Acinetobacter baumannii This patent atpD 244 Acinetobacter
lwoffii This patent atpD 245 Staphylococcus saprophyticus This
patent atpD 246 Alcaligenes faecalis subsp. faecalis This patent
atpD 247 Bacillus anthracis This patent atpD 248 Bacillus cereus
This patent atpD 249 Bacteroides distasonis This patent atpD 250
Bacteroides ovatus This patent atpD 251 Leclercia adecarboxylata
This patent atpD 252 Stenotrophomonas maltophilia This patent atpD
253 Bartonella henselae This patent atpD 254 Bifidobacterium
adolescentis This patent atpD 255 Brucella abortus This patent atpD
256 Cedecea davisae This patent atpD 257 Cedecea lapagei This
patent atpD 258 Cedecea neteri This patent atpD 259
Chryseobacterium meningosepticum This patent atpD 260 Citrobacter
amalonaticus This patent atpD 261 Citrobacter braakii This patent
atpD 262 Citrobacter koseri This patent atpD 263 Citrobacter
farmeri This patent atpD 264 Citrobacter freundii This patent atpD
265 Citrobacter koseri This patent atpD 266 Citrobacter sedlakii
This patent atpD 267 Citrobacter werkmanii This patent atpD 268
Citrobacter youngae This patent atpD 269 Clostridium innocuum This
patent atpD 270 Clostridium perfringens This patent atpD 272
Corynebacterium diphtheriae This patent atpD 273 Corynebacterium
pseudodiphtheriticum This patent atpD 274 Corynebacterium ulcerans
This patent atpD 275 Corynebacterium urealyticum This patent atpD
276 Coxiella burnetii This patent atpD 277 Edwardsiella hoshinae
This patent atpD 278 Edwardsiella tarda This patent atpD 279
Eikenella corrodens This patent atpD 280 Enterobacter agglomerans
This patent atpD 281 Enterobacter arnnigenus This patent atpD 282
Enterobacter asburiae This patent atpD 283 Enterobacter
cancerogenus This patent atpD 284 Enterobacter cloacae This patent
atpD 285 Enterobacter gergoviae This patent atpD 286 Enterobacter
hormaechei This patent atpD 287 Enterobacter sakazakii This patent
atpD 288 Enterococcus avium This patent atpD 289 Enterococcus
casseliflavus This patent atpD 290 Enterococcus durans This patent
atpD 291 Enterococcus faecalis This patent atpD 292 Enterococcus
faecium This patent atpD 293 Enterococcus gallinarum This patent
atpD 294 Enterococcus saccharolyticus This patent atpD 295
Escherichia fergusonii This patent atpD 296 Escherichia hermannii
This patent atpD 297 Escherichia vulneris This patent atpD 298
Eubacterium lentum This patent atpD 299 Ewingella americana This
patent atpD 300 Francisella tularensis This patent atpD 301
Fusobacterium gonidiaformans This patent atpD 302 Fusobacterium
necrophorum subsp. necrophorum This patent atpD 303 Fusobacterium
nucleatum subsp. polymorphum This patent atpD 304 Gardnerella
vaginalis This patent atpD 305 Gemella haemolysans This patent atpD
306 Gemella morbillorum This patent atpD 307 Haemophilus ducreyi
This patent atpD 308 Haemophilus haemolyticus This patent atpD 309
Haemophilus parahaemolyticus This patent atpD 310 Haemophilus
parainfluenzae This patent atpD 311 Hafnia alvei This patent atpD
312 Kingella kingae This patent atpD 313 Klebsiella pneumoniae
subsp. ozaenae This patent atpD 314 Klebsiella omithinolytica This
patent atpD 315 Klebsiella oxytoca This patent atpD 316 Klebsiella
planticola This patent atpD 317 Klebsiella pneumoniae subsp.
pneumoniae This patent atpD 318 Kluyvera ascorbata This patent atpD
319 Kluyvera cryocrescens This patent atpD 320 Kluyvera georgiana
This patent atpD 321 Lactobacillus acidophilus This patent atpD 322
Legionella pneumophila subsp. pneumophila This patent atpD 323
Leminorella grimontii This patent atpD 324 Listeria monocytogenes
This patent atpD 325 Micrococcus lylae This patent atpD 326
Moellerella wisconsensis This patent atpD 327 Moraxella catarrhalis
This patent atpD 328 Moraxella osloensis This patent atpD 329
Morganella morganii subsp. morganii This patent atpD 330 Pantoea
agglomerans This patent atpD 331 Pantoea dispersa This patent atpD
332 Pasteurella multocida This patent atpD 333 Pragia fontium This
patent atpD 334 Proteus mirabilis This patent atpD 335 Proteus
vulgaris This patent atpD 336 Providencia alcalifaciens This patent
atpD 337 Providencia rettgeri This patent atpD 338 Providencia
rustigianii This patent atpD 339 Providencia stuartii This patent
atpD 340 Psychrobacter phenylpyruvicum This patent atpD 341
Rahnella aquatilis This patent atpD 342 Salmonella choleraesuis
subsp. arizonae This patent atpD 343 Salmonella choleraesuis subsp.
choleraesuis This patent atpD serotype Choleraesuis 344 Salmonella
choleraesuis subsp. diarizonae This patent atpD 345 Salmonella
choleraesuis subsp. houtenae This patent atpD 346 Salmonella
choleraesuis subsp. indica This patent atpD 347 Salmonella
choleraesuis subsp. choleraesuis This patent atpD serotype
Paratyphi A 348 Salmonella choleraesuis subsp. choleraesuis This
patent atpD serotype Paratyphi B 349 Salmonella choleraesuis subsp.
salamae This patent atpD 350 Salmonella choleraesuis subsp.
choleraesuis serotype Typhi This patent atpD 351 Salmonella
choleraesuis subsp. choleraesuis This patent atpD serotype
Typhimurium 352 Salmonella choleraesuis subsp. choleraesuis This
patent atpD serotype Virchow 353 Serratia ficaria This patent atpD
354 Serratia fonticola This patent atpD 355 Serratia grimesii This
patent atpD 356 Serratia liquefaciens This patent atpD 357 Serratia
marcescens This patent atpD 358 Serratia odorifera This patent atpD
359 Serratia plymuthica This patent atpD 360 Serratia rubidaea This
patent atpD 361 Pseudomonas putida This patent atpD 362 Shigella
boydii This patent atpD 363 Shigella dysenteriae This patent atpD
364 Shigella flexneri This patent atpD 365 Shigella sonnei This
patent atpD 366 Staphylococcus aureus This patent atpD 367
Staphylococcus auricularis This patent atpD 368 Staphylococcus
capitis subsp. capitis This patent atpD 369 Staphylococcus cohnii
subsp. cohnii This patent atpD 370 Staphylococcus epidermidis This
patent atpD 371 Staphylococcus haemolyticus This patent atpD 372
Staphylococcus hominis subsp. hominis This patent atpD 373
Staphylococcus hominis This patent atpD 374 Staphylococcus
lugdunensis This patent atpD 375 Staphylococcus saprophyticus This
patent atpD 376 Staphylococcus simulans This patent atpD 377
Staphylococcus wameri This patent atpD 378 Streptococcus
acidominimus This patent atpD 379 Streptococcus agalactiae This
patent atpD 380 Streptococcus agalactiae This patent atpD 381
Streptococcus agalactiae This patent atpD 382 Streptococcus
agalactiae This patent atpD 383 Streptococcus agalactiae This
patent atpD 384 Streptococcus dysgalactiae This patent atpD 385
Streptococcus equi subsp. equi This patent atpD 386 Streptococcus
anginosus This patent atpD 387 Streptococcus salivarius This patent
atpD 388 Streptococcus suis This patent atpD 389 Streptococcus
uberis This patent atpD 390 Tatumella ptyseos This patent atpD 391
Trabulsiella guamensis This patent atpD 392 Yersinia bercovieri
This patent atpD 393 Yersinia enterocolitica This patent atpD 394
Yersinia frederiksenii This patent atpD 395 Yersinia intermedia
This patent atpD 396 Yersinia pseudotuberculosis This patent atpD
397 Yersinia rohdei This patent atpD 398 Yokenella regensburgei
This patent atpD 399 Yarrowia lipolytica This patent tuf (EF-1) 400
Absidia corymbifera This patent tuf (EF-1) 401 Alternaria alternata
This patent tuf (EF-1) 402 Aspergillus flavus This patent tuf
(EF-1) 403 Aspergillus fumigatus This patent tuf (EF-1) 404
Aspergillus fumigatus This patent tuf (EF-1) 405 Aspergillus niger
This patent tuf (EF-1) 406 Blastoschizomyces capitatus This patent
tuf (EF-1) 407 Candida albicans This patent tuf (EF-1) 408 Candida
albicans This patent tuf (EF-1) 409 Candida albicans This patent
tuf (EF-1) 410 Candida albicans This patent tuf (EF-1) 411 Candida
albicans This patent tuf (EF-1) 412 Candida dubliniensis This
patent tuf (EF-1) 413 Candida catenulata This patent tuf (EF-1) 414
Candida dubliniensis This patent tuf (EF-1) 415 Candida
dubliniensis This patent tuf (EF-1) 416 Candida famata This patent
tuf (EF-1) 417 Candida glabrata WO98/20157 tuf (EF-1) 418 Candida
guilliermondii This patent tuf (EF-1) 419 Candida haemulonii This
patent tuf (EF-1) 420 Candida inconspicua This patent tuf (EF-1)
421 Candida kefyr This patent tuf (EF-1) 422 Candida krusei
WO98/20157 tuf (EF-1) 423 Candida lambica This patent tuf (EF-1)
424 Candida lusitaniae This patent tuf (EF-1) 425 Candida
norvegensis This patent tuf (EF-1) 426 Candida parapsilosis
WO98/20157 tuf (EF-1) 427 Candida rugosa This patent tuf (EF-1) 428
Candida sphaerica This patent tuf (EF-1) 429 Candida tropicalis
WO98/20157 tuf (EF-1) 430 Candida utilis This patent tuf (EF-1) 431
Candida viswanathii This patent tuf (EF-1) 432 Candida zeylanoides
This patent tuf (EF-1) 433 Coccidioides immitis This patent tuf
(EF-1) 434 Cryptococcus albidus This patent tuf (EF-1) 435
Exophiala jeanselmei This patent tuf (EF-1) 436 Fusarium oxysporum
This patent tuf (EF-1) 437 Geotrichum sp. This patent tuf (EF-1)
438 Histoplasma capsulatum This patent tuf (EF-1) 439 Issatchenkia
orientalis Kudrjanzev This patent tuf (EF-1) 440 Malassezia furfur
This patent tuf (EF-1) 441 Malassezia pachydermatis This patent tuf
(EF-1) 442 Malbranchea filamentosa This patent tuf (EF-1) 443
Metschnikowia pulcherrima This patent tuf (EF-1) 444 Paecilomyces
lilacinus This patent tuf (EF-1) 445 Paracoccidioides brasiliensis
This patent tuf (EF-1) 446 Penicillium marneffei This patent tuf
(EF-1) 447 Pichia anomala This patent tuf (EF-1) 448 Pichia anomala
This patent tuf (EF-1) 449 Pseudallescheria boydii This patent tuf
(EF-1) 450 Rhizopus oryzae This patent tuf (EF-1) 451 Rhodotorula
minuta This patent tuf (EF-1) 452 Sporobolomyces salmonicolor This
patent tuf (EF-1) 453 Sporothrix schenckii This patent tuf (EF-1)
454 Stephanoascus ciferrii This patent tuf (EF-1) 455 Trichophyton
mentagrophytes This patent tuf (EF-1) 456 Trichosporon cutaneum
This patent tuf (EF-1) 457 Wangiella dermatitidis This patent tuf
(EF-1) 458 Aspergillus fumigatus This patent atpD 459
Blastoschizomyces capitatus This patent atpD 460 Candida albicans
This patent atpD 461 Candida dubliniensis This patent atpD 462
Candida famata This patent atpD 463 Candida glabrata This patent
atpD 464 Candida guilliermondii This patent atpD 465 Candida
haemulonii This patent atpD 466 Candida inconspicua This patent
atpD 467 Candida kefyr This patent atpD 468 Candida krusei This
patent atpD 469 Candida lambica This patent atpD 470 Candida
lusitaniae This patent atpD 471 Candida norvegensis This patent
atpD 472 Candida parapsilosis This patent atpD 473 Candida rugosa
This patent atpD 474 Candida sphaerica This patent atpD 475 Candida
tropicalis This patent atpD 476 Candida utilis This patent atpD 477
Candida viswanathii This patent atpD 478 Candida zeylanoides This
patent atpD 479 Coccidioides immitis This patent atpD 480
Cryptococcus albidus This patent atpD 481 Fusarium oxysporum This
patent atpD 482 Geotrichum sp. This patent atpD 483 Histoplasma
capsulatum This patent atpD 484 Malassezia furfur This patent atpD
485 Malassezia pachydermatis This patent atpD 486 Metschnikowia
pulcherrima This patent atpD 487 Penicillium mameffei This patent
atpD
488 Pichia anomala This patent atpD 489 Pichia anomala This patent
atpD 490 Rhodotorula minuta This patent atpD 491 Rhodotorula
mucilaginosa This patent atpD 492 Sporobolomyces salmonicolor This
patent atpD 493 Sporothrix schenckii This patent atpD 494
Stephanoascus ciferrii This patent atpD 495 Trichophyton
mentagrophytes This patent atpD 496 Wangiella dermatitidis This
patent atpD 497 Yarrowia lipolytica This patent atpD 498
Aspergillus fumigatus This patent tuf (M) 499 Blastoschizomyces
capitatus This patent tuf (M) 500 Candida rugosa This patent tuf
(M) 501 Coccidioides immitis This patent tuf (M) 502 Fusarium
oxysporum This patent tuf (M) 503 Histoplasma capsulatum This
patent tuf (M) 504 Paracoccidioides brasiliensis This patent tuf
(M) 505 Penicillium marneffei This patent tuf (M) 506 Pichia
anomala This patent tuf (M) 507 Trichophyton mentagrophytes This
patent tuf (M) 508 Yarrowia lipolytica This patent tuf (M) 509
Babesia bigemina This patent tuf (EF-1) 510 Babesia bovis This
patent tuf (EF-1) 511 Crithidia fasciculata This patent tuf (EF-1)
512 Entamoeba histolytica This patent tuf (EF-1) 513 Giardia
lamblia This patent tuf (EF-1) 514 Leishmania tropica This patent
tuf (EF-1) 515 Leishmania aethiopica This patent tuf (EF-1) 516
Leishmania tropica This patent tuf (EF-1) 517 Leishmania donovani
This patent tuf (EF-1) 518 Leishmania infantum This patent tuf
(EF-1) 519 Leishmania enriettii This patent tuf (EF-1) 520
Leishmania gerbilli This patent tuf (EF-1) 521 Leishmania hertigi
This patent tuf (EF-1) 522 Leishmania major This patent tuf (EF-1)
523 Leishmania amazonensis This patent tuf (EF-1) 524 Leishmania
mexicana This patent tuf (EF-1) 525 Leishmania tarentolae This
patent tuf (EF-1) 526 Leishmania tropica This patent tuf (EF-1) 527
Neospora caninum This patent tuf (EF-1) 528 Trichomonas vaginalis
This patent tuf (EF-1) 529 Trypanosoma brucei subsp. brucei This
patent tuf (EF-1) 530 Crithidia fasciculata This patent atpD 531
Leishmania tropica This patent atpD 532 Leishmania aethiopica This
patent atpD 533 Leishmania donovani This patent atpD 534 Leishmania
infantum This patent atpD 535 Leishmania gerbilli This patent atpD
536 Leishmania hertigi This patent atpD 537 Leishmania major This
patent atpD 538 Leishmania amazonensis This patent atpD 607
Enterococcus faecalis WO98/20157 tuf 608 Enterococcus faecium
WO98/20157 tuf 609 Enterococcus gallinarum WO98/20157 tuf 610
Haemophilus influenzae WO98/20157 tuf 611 Staphylococcus
epidermidis WO98/20157 tuf 612 Salmonella choleraesuis subsp.
choleraesuis This patent tuf serotype Paratyphi A 613 Serratia
ficaria This patent tuf 614 Enterococcus malodoratus This patent
tuf (C) 615 Enterococcus durans This patent tuf (C) 616
Enterococcus pseudoavium This patent tuf (C) 617 Enterococcus
dispar This patent tuf (C) 618 Enterococcus avium This patent tuf
(C) 619 Saccharomyces cerevisiae Database tuf (M) 621 Enterococcus
faecium This patent tuf (C) 622 Saccharomyces cerevisiae This
patent tuf (EF-1) 623 Cryptococcus neoformans This patent tuf
(EF-1) 624 Candida albicans WO98/20157 tuf (EF-1) 662
Corynebacterium diphtheriae WO98/20157 tuf 663 Candida catenulata
This patent atpD 665 Saccharomyces cerevisiae Database tuf (EF-1)
666 Saccharomyces cerevisiae Database atpD 667 Trypanosoma cruzi
This patent atpD 668 Corynebacterium glutamicum Database tuf 669
Escherichia coli Database atpD 670 Helicobacter pylori Database
atpD 671 Clostridium acetobutylicum Database atpD 672 Cytophaga
lytica Database atpD 673 Ehrlichia risticii This patent atpD 674
Vibrio cholerae This patent atpD 675 Vibrio cholerae This patent
tuf 676 Leishmania enriettii This patent atpD 677 Babesia microti
This patent tuf (EF-1) 678 Cryptococcus neoformans This patent atpD
679 Cryptococcus neoformans This patent atpD 680 Cunninghamella
bertholletiae This patent atpD 684 Candida tropicalis Database atpD
(V) 685 Enterococcus hirae Database atpD (V) 686 Chlamydia
pneumoniae Database atpD (V) 687 Halobacterium salinarum Database
atpD (V) 688 Homo sapiens Database atpD (V) 689 Plasmodium
falciparum Database atpD (V) 690 Saccharomyces cerevisiae Database
atpD (V) 691 Schizosaccharomyces pombe Database atpD (V) 692
Trypanosoma congolense Database atpD (V) 693 Thermus thermophilus
Database atpD (V) 698 Escherichia coli WO98/20157 tuf 709 Borrelia
burgdorferi Database atpD (V) 710 Treponema pallidum Database atpD
(V) 711 Chlamydia trachomatis Genome project atpD (V) 712
Enterococcus faecalis Genome project atpD (V) 713 Methanosarcina
barkeri Database atpD (V) 714 Methanococcus jannaschii Database
atpD (V) 715 Porphyromonas gingivalis Genome project atpD (V) 716
Streptococcus pneumoniae Genome project atpD (V) 717 Burkholderia
mallei This patent tuf 718 Burkholderia pseudomallei This patent
tuf 719 Clostridium beijerinckii This patent tuf 720 Clostridium
innocuum This patent tuf 721 Clostridium novyi This patent tuf 722
Clostridium septicum This patent tuf 723 Clostridium tertium This
patent tuf 724 Clostridium tetani This patent tuf 725 Enterococcus
malodoratus This patent tuf 726 Enterococcus sulfureus This patent
tuf 727 Lactococcus garvieae This patent tuf 728 Mycoplasma pirum
This patent tuf 729 Mycoplasma salivarium This patent tuf 730
Neisseria polysaccharea This patent tuf 731 Salmonella choleraesuis
subsp. choleraesuis This patent tuf serotype Enteritidis 732
Salmonella choleraesuis subsp. choleraesuis This patent tuf
serotype Gallinarum 733 Salmonella choleraesuis subsp. choleraesuis
This patent tuf serotype Paratyphi B 734 Salmonella choleraesuis
subsp. choleraesuis This patent tuf serotype Virchow 735 Serratia
grimesii This patent tuf 736 Clostridium difficile This patent tuf
737 Burkholderia pseudomallei This patent atpD 738 Clostridium
bifermentans This patent atpD 739 Clostridium beijerinckii This
patent atpD 740 Clostridium difficile This patent atpD 741
Clostridium ramosum This patent atpD 742 Clostridium septicum This
patent atpD 743 Clostridium tertium This patent atpD 744 Comamonas
acidovorans This patent atpD 745 Klebsiella pneumoniae subsp.
rhinoscleromatis This patent atpD 746 Neisseria canis This patent
atpD 747 Neisseria cinerea This patent atpD 748 Neisseria cuniculi
This patent atpD 749 Neisseria elongata subsp. elongata This patent
atpD 750 Neisseria flavescens This patent atpD 751 Neisseria
gonorrhoeae This patent atpD 752 Neisseria gonorrhoeae This patent
atpD 753 Neisseria lactamica This patent atpD 754 Neisseria
meningitidis This patent atpD 755 Neisseria mucosa This patent atpD
756 Neisseria subflava This patent atpD 757 Neisseria weaveri This
patent atpD 758 Neisseria animalis This patent atpD 759 Proteus
penneri This patent atpD 760 Salmonella choleraesuis subsp.
choleraesuis This patent atpD serotype Enteritidis 761 Yersinia
pestis This patent atpD 762 Burkholderia mallei This patent atpD
763 Clostridium sordellii This patent atpD 764 Clostridium novyi
This patent atpD 765 Clostridium botulinum This patent atpD 766
Clostridium histolyticum This patent atpD 767 Peptostreptococcus
prevotii This patent atpD 768 Absidia corymbifera This patent atpD
769 Alternaria alternata This patent atpD 770 Aspergillus flavus
This patent atpD 771 Mucor circinelloides This patent atpD 772
Piedraia hortai This patent atpD 773 Pseudallescheria boydii This
patent atpD 774 Rhizopus oryzae This patent atpD 775 Scopulariopsis
koningii This patent atpD 776 Trichophyton mentagrophytes This
patent atpD 777 Trichophyton tonsurans This patent atpD 778
Trichosporon cutaneum This patent atpD 779 Cladophialophora
carrionii This patent tuf (EF-1) 780 Cunninghamella bertholletiae
This patent tuf (EF-1) 781 Curvularia lunata This patent tuf (EF-1)
782 Fonsecaea pedrosoi This patent tuf (EF-1) 783 Microsporum
audouinii This patent tuf (EF-1) 784 Mucor circinelloides This
patent tuf (EF-1) 785 Phialophora verrucosa This patent tuf (EF-1)
786 Saksenaea vasiformis This patent tuf (EF-1) 787 Syncephalastrum
racemosum This patent tuf (EF-1) 788 Trichophyton tonsurans This
patent tuf (EF-1) 789 Trichophyton mentagrophytes This patent tuf
(EF-1) 790 Bipolaris hawaiiensis This patent tuf (EF-1) 791
Aspergillus fumigatus This patent tuf (M) 792 Trichophyton
mentagrophytes This patent tuf (M) 827 Clostridium novyi This
patent atpD (V) 828 Clostridium difficile This patent atpD (V) 829
Clostridium septicum This patent atpD (V) 830 Clostridium botulinum
This patent atpD (V) 831 Clostridium perfringens This patent atpD
(V) 832 Clostridium tetani This patent atpD (V) 833 Streptococcus
pyogenes Database atpD (V) 834 Babesia bovis This patent atpD (V)
835 Cryptosporidium parvum This patent atpD (V) 836 Leishmania
infantum This patent atpD (V) 837 Leishmania major This patent atpD
(V) 838 Leishmania tarentolae This patent atpD (V) 839 Trypanosoma
brucei This patent atpD (V) 840 Trypanosoma cruzi This patent tuf
(EF-1) 841 Trypanosoma cruzi This patent tuf (EF-1) 842 Trypanosoma
cruzi This patent tuf (EF-1) 843 Babesia bovis This patent tuf (M)
844 Leishmania aethiopica This patent tuf (M) 845 Leishmania
amazonensis This patent tuf (M) 846 Leishmania donovani This patent
tuf (M) 847 Leishmania infantum This patent tuf (M) 848 Leishmania
enriettii This patent tuf (M) 849 Leishmania gerbilli This patent
tuf (M) 850 Leishmania major This patent tuf (M) 851 Leishmania
mexicana This patent tuf (M) 852 Leishmania tarentolae This patent
tuf (M) 853 Trypanosoma cruzi This patent tuf (M) 854 Trypanosoma
cruzi This patent tuf (M) 855 Trypanosoma cruzi This patent tuf (M)
856 Babesia bigemina This patent atpD 857 Babesia bovis This patent
atpD 858 Babesia microti This patent atpD 859 Leishmania guyanensis
This patent atpD 860 Leishmania mexicana This patent atpD 861
Leishmania tropica This patent atpD 862 Leishmania tropica This
patent atpD 863 Bordetella pertussis Database tuf 864 Trypanosoma
brucei brucei Database tuf (EF-1) 865 Cryptosporidium parvum This
patent tuf (EF-1) 866 Staphylococcus saprophyticus This patent atpD
867 Zoogloea ramigera This patent atpD 868 Staphylococcus
saprophyticus This patent tuf 869 Enterococcus casseliflavus This
patent tuf 870 Enterococcus casseliflavus This patent tuf 871
Enterococcus flavescens This patent tuf 872 Enterococcus gallinarum
This patent tuf 873 Enterococcus gallinarum This patent tuf 874
Staphylococcus haemolyticus This patent tuf 875 Staphylococcus
epidermidis This patent tuf 876 Staphylococcus epidermidis This
patent tuf 877 Staphylococcus epidermidis This patent tuf 878
Staphylococcus epidermidis This patent tuf 879 Enterococcus
gallinarum This patent tuf 880 Pseudomonas aeruginosa This patent
tuf 881 Enterococcus casseliflavus This patent tuf 882 Enterococcus
casseliflavus This patent tuf 883 Enterococcus faecalis This patent
tuf 884 Enterococcus faecalis This patent tuf 885 Enterococcus
faecium This patent tuf 886 Enterococcus faecium This patent tuf
887 Zoogloea ramigera This patent tuf 888 Enterococcus faecalis
This patent tuf 889 Aspergillus fumigatus This patent atpD 890
Penicillium mameffei This patent atpD
891 Paecilomyces lilacinus This patent atpD 892 Penicillium
mameffei This patent atpD 893 Sporothrix schenckii This patent atpD
894 Malbranchea filamentosa This patent atpD 895 Paecilomyces
lilacinus This patent atpD 896 Aspergillus niger This patent atpD
897 Aspergillus fumigatus This patent tuf (EF-1) 898 Penicillium
mameffei This patent tuf (EF-1) 899 Piedraia hortai This patent tuf
(EF-1) 900 Paecilomyces lilacinus This patent tuf (EF-1) 901
Paracoccidioides brasiliensis This patent tuf (EF-1) 902 Sporothrix
schenckii This patent tuf (EF-1) 903 Penicillium mameffei This
patent tuf (EF-1) 904 Curvularia lunata This patent tuf (M) 905
Aspergillus niger This patent tuf (M) 906 Bipolaris hawaiiensis
This patent tuf (M) 907 Aspergillus flavus This patent tuf (M) 908
Alternaria alternata This patent tuf (M) 909 Penicillium mameffei
This patent tuf (M) 910 Penicillium mameffei This patent tuf (M)
918 Escherichia coli Database recA 929 Bacteroides fragilis This
patent atpD (V) 930 Bacteroides distasonis This patent atpD (V) 931
Porphyromonas asaccharolytica This patent atpD (V) 932 Listeria
monocytogenes This patent tuf 939 Saccharomyces cerevisiae Database
recA (Rad51) 940 Saccharomyces cerevisiae Database recA (Dmc1) 941
Cryptococcus humicolus This patent atpD 942 Escherichia coli This
patent atpD 943 Escherichia coli This patent atpD 944 Escherichia
coli This patent atpD 945 Escherichia coli This patent atpD 946
Neisseria polysaccharea This patent atpD 947 Neisseria sicca This
patent atpD 948 Streptococcus mitis This patent atpD 949
Streptococcus mitis This patent atpD 950 Streptococcus mitis This
patent atpD 951 Streptococcus oralis This patent atpD 952
Streptococcus pneumoniae This patent atpD 953 Streptococcus
pneumoniae This patent atpD 954 Streptococcus pneumoniae This
patent atpD 955 Streptococcus pneumoniae This patent atpD 956
Babesia microti This patent atpD (V) 957 Entamoeba histolytica This
patent atpD (V) 958 Fusobacterium nucleatum subsp. polymorphum This
patent atpD (V) 959 Leishmania aethiopica This patent atpD (V) 960
Leishmania tropica This patent atpD (V) 961 Leishmania guyanensis
This patent atpD (V) 962 Leishmania donovani This patent atpD (V)
963 Leishmania hertigi This patent atpD (V) 964 Leishmania mexicana
This patent atpD (V) 965 Leishmania tropica This patent atpD (V)
966 Peptostreptococcus anaerobius This patent atpD (V) 967
Bordetella pertussis This patent tuf 968 Bordetella pertussis This
patent tuf 969 Enterococcus columbae This patent tuf 970
Enterococcus flavescens This patent tuf 971 Streptococcus
pneumoniae This patent tuf 972 Escherichia coli This patent tuf 973
Escherichia coli This patent tuf 974 Escherichia coli This patent
tuf 975 Escherichia coli This patent tuf 976 Mycobacterium avium
This patent tuf 977 Streptococcus pneumoniae This patent tuf 978
Mycobacterium gordonae This patent tuf 979 Streptococcus pneumoniae
This patent tuf 980 Mycobacterium tuberculosis This patent tuf 981
Staphylococcus warneri This patent tuf 982 Streptococcus mitis This
patent tuf 983 Streptococcus mitis This patent tuf 984
Streptococcus mitis This patent tuf 985 Streptococcus oralis This
patent tuf 986 Streptococcus pneumoniae This patent tuf 987
Enterococcus hirae This patent tuf (C) 988 Enterococcus mundtii
This patent tuf (C) 989 Enterococcus raffinosus This patent tuf (C)
990 Bacillus anthracis This patent recA 991 Prevotella
melaninogenica This patent recA 992 Enterococcus casseliflavus This
patent tuf 993 Streptococcus pyogenes Database speA 1002
Streptococcus pyogenes WO98/20157 tuf 1003 Bacillus cereus This
patent recA 1004 Streptococcus pneumoniae This patent pbp1a 1005
Streptococcus pneumoniae This patent pbp1a 1006 Streptococcus
pneumoniae This patent pbp1a 1007 Streptococcus pneumoniae This
patent pbp1a 1008 Streptococcus pneumoniae This patent pbp1a 1009
Streptococcus pneumoniae This patent pbp1a 1010 Streptococcus
pneumoniae This patent pbp1a 1011 Streptococcus pneumoniae This
patent pbp1a 1012 Streptococcus pneumoniae This patent pbp1a 1013
Streptococcus pneumoniae This patent pbp1a 1014 Streptococcus
pneumoniae This patent pbp1a 1015 Streptococcus pneumoniae This
patent pbp1a 1016 Streptococcus pneumoniae This patent pbp1a 1017
Streptococcus pneumoniae This patent pbp1a 1018 Streptococcus
pneumoniae This patent pbp1a 1019 Streptococcus pneumoniae This
patent pbp2b 1020 Streptococcus pneumoniae This patent pbp2b 1021
Streptococcus pneumoniae This patent pbp2b 1022 Streptococcus
pneumoniae This patent pbp2b 1023 Streptococcus pneumoniae This
patent pbp2b 1024 Streptococcus pneumoniae This patent pbp2b 1025
Streptococcus pneumoniae This patent pbp2b 1026 Streptococcus
pneumoniae This patent pbp2b 1027 Streptococcus pneumoniae This
patent pbp2b 1028 Streptococcus pneumoniae This patent pbp2b 1029
Streptococcus pneumoniae This patent pbp2b 1030 Streptococcus
pneumoniae This patent pbp2b 1031 Streptococcus pneumoniae This
patent pbp2b 1032 Streptococcus pneumoniae This patent pbp2b 1033
Streptococcus pneumoniae This patent pbp2b 1034 Streptococcus
pneumoniae This patent pbp2x 1035 Streptococcus pneumoniae This
patent pbp2x 1036 Streptococcus pneumoniae This patent pbp2x 1037
Streptococcus pneumoniae This patent pbp2x 1038 Streptococcus
pneumoniae This patent pbp2x 1039 Streptococcus pneumoniae This
patent pbp2x 1040 Streptococcus pneumoniae This patent pbp2x 1041
Streptococcus pneumoniae This patent pbp2x 1042 Streptococcus
pneumoniae This patent pbp2x 1043 Streptococcus pneumoniae This
patent pbp2x 1044 Streptococcus pneumoniae This patent pbp2x 1045
Streptococcus pneumoniae This patent pbp2x 1046 Streptococcus
pneumoniae This patent pbp2x 1047 Streptococcus pneumoniae This
patent pbp2x 1048 Streptococcus pneumoniae This patent pbp2x 1049
Enterococcus faecium This patent vanA 1050 Enterococcus gallinarum
This patent vanA 1051 Enterococcus faecium This patent vanA 1052
Enterococcus faecium This patent vanA 1053 Enterococcus faecium
This patent vanA 1054 Enterococcus faecalis This patent vanA 1055
Enterococcus gallinarum This patent vanA 1056 Enterococcus faecium
This patent vanA 1057 Enterococcus flavescens This patent vanA 1058
Enterococcus gallinarum This patent vanC1 1059 Enterococcus
gallinarum This patent vanC1 1060 Enterococcus casseliflavus This
patent vanC2 1061 Enterococcus casseliflavus This patent vanC2 1062
Enterococcus casseliflavus This patent vanC2 1063 Enterococcus
casseliflavus This patent vanC2 1064 Enterococcus flavescens This
patent vanC3 1065 Enterococcus flavescens This patent vanC3 1066
Enterococcus flavescens This patent vanC3 1067 Enterococcus faecium
This patent vanXY 1068 Enterococcus faecium This patent vanXY 1069
Enterococcus faecium This patent vanXY 1070 Enterococcus faecalis
This patent vanXY 1071 Enterococcus gallinarum This patent vanXY
1072 Enterococcus faecium This patent vanXY 1073 Enterococcus
flavescens This patent vanXY 1074 Enterococcus faecium This patent
vanXY 1075 Enterococcus gallinarum This patent vanXY 1076
Escherichia coli Database stx.sub.1 1077 Escherichia coli Database
stx.sub.2 1093 Staphylococcus saprophyticus This patent unknown
1117 Enterococcus faecium Database vanB 1138 Enterococcus
gallinarum Database vanC1 1139 Enterococcus faecium Database vanA
1140 Enterococcus casseliflavus Database vanC2 1141 Enterococcus
faecium Database vanHAXY 1169 Streptococcus pneumoniae Database
pbp1a 1172 Streptococcus pneumoniae Database pbp2b 1173
Streptococcus pneumoniae Database pbp2x 1178 Staphylococcus aureus
Database mecA 1183 Streptococcus pneumoniae Database hexA 1184
Streptococcus pneumoniae This patent hexA 1185 Streptococcus
pneumoniae This patent hexA 1186 Streptococcus pneumoniae This
patent hexA 1187 Streptococcus pneumoniae This patent hexA 1188
Streptococcus oralis This patent hexA 1189 Streptococcus mitis This
patent hexA 1190 Streptococcus mitis This patent hexA 1191
Streptococcus mitis This patent hexA 1198 Staphylococcus
saprophyticus This patent unknown 1215 Streptococcus pyogenes
Database pcp 1230 Escherichia coli Database tuf (EF-G) 1242
Enterococcus faecium Database ddl 1243 Enterococcus faecalis
Database mtlF, mtlD 1244 Staphylococcus aureus subsp. aureus This
patent unknown 1245 Bacillus anthracis This patent atpD 1246
Bacillus mycoides This patent atpD 1247 Bacillus thuringiensis This
patent atpD 1248 Bacillus thuringiensis This patent atpD 1249
Bacillus thuringiensis This patent atpD 1250 Bacillus
weihenstephanensis This patent atpD 1251 Bacillus thuringiensis
This patent atpD 1252 Bacillus thuringiensis This patent atpD 1253
Bacillus cereus This patent atpD 1254 Bacillus cereus This patent
atpD 1255 Staphylococcus aureus This patent gyrA 1256 Bacillus
weihenstephanensis This patent atpD 1257 Bacillus anthracis This
patent atpD 1258 Bacillus thuringiensis This patent atpD 1259
Bacillus cereus This patent atpD 1260 Bacillus cereus This patent
atpD 1261 Bacillus thuringiensis This patent atpD 1262 Bacillus
thuringiensis This patent atpD 1263 Bacillus thuringiensis This
patent atpD 1264 Bacillus thuringiensis This patent atpD 1265
Bacillus anthracis This patent atpD 1266 Paracoccidioides
brasiliensis This patent tuf (EF-1) 1267 Blastomyces dermatitidis
This patent tuf (EF-1) 1268 Histoplasma capsulatum This patent tuf
(EF-1) 1269 Trichophyton rubrum This patent tuf (EF-1) 1270
Microsporum canis This patent tuf (EF-1) 1271 Aspergillus
versicolor This patent tuf (EF-1) 1272 Exophiala moniliae This
patent tuf (EF-1) 1273 Hortaea wemeckii This patent tuf (EF-1) 1274
Fusarium solani This patent tuf (EF-1) 1275 Aureobasidium pullulans
This patent tuf (EF-1) 1276 Blastomyces dermatitidis This patent
tuf (EF-1) 1277 Exophiala dermatitidis This patent tuf (EF-1) 1278
Fusarium moniliforme This patent tuf (EF-1) 1279 Aspergillus
terreus This patent tuf (EF-1) 1280 Aspergillus fumigatus This
patent tuf (EF-1) 1281 Cryptococcus laurentii This patent tuf
(EF-1) 1282 Emmonsia parva This patent tuf (EF-1) 1283 Fusarium
solani This patent tuf (EF-1) 1284 Sporothrix schenckii This patent
tuf (EF-1) 1285 Aspergillus nidulans This patent tuf (EF-1) 1286
Cladophialophora carrionii This patent tuf (EF-1) 1287 Exserohilum
rostratum This patent tuf (EF-1) 1288 Bacillus thuringiensis This
patent recA 1289 Bacillus thuringiensis This patent recA 1299
Staphylococcus aureus Database gyrA 1300 Escherichia coli Database
gyrA 1307 Staphylococcus aureus Database gyrB 1320 Escherichia coli
Database parC (grlA) 1321 Staphylococcus aureus Database parC
(grlA) 1328 Staphylococcus aureus Database parE (grlB) 1348
unidentified bacterium Database aac2Ia 1351 Pseudomonas aeruginosa
Database aac3Ib 1356 Serratia marcescens Database aac3IIb 1361
Escherichia coli Database aac3IVa 1366 Enterobacter cloacae
Database aac3VIa 1371 Citrobacter koseri Database aac6Ia 1376
Serratia marcescens Database aac6Ic 1381 Escherichia coli Database
ant3Ia 1386 Staphylococcus aureus Database ant4Ia 1391 Escherichia
coli Database aph3Ia 1396 Escherichia coli Database aph3IIa 1401
Enterococcus faecalis Database aph3IIIa 1406 Acinetobacter
baumannii Database aph3VIa 1411 Pseudomonas aeruginosa Database
blaCARB 1416 Klebsiella pneumoniae Database blaCMY-2 1423
Escherichia coli Database blaCTX-M-1 1428 Salmonella choleraesuis
subsp. choleraesuis serotype Database blaCTX-M-2 Typhimurium
1433 Pseudomonas aeruginosa Database blaIMP 1438 Escherichia coli
Database blaOXA2 1439 Pseudomonas aeruginosa Database blaOXA10 1442
Pseudomonas aeruginosa Database blaPER1 1445 Salmonella
choleraesuis subsp. choleraesuis serotype Database blaPER2
Typhimurium 1452 Staphylococcus epidermidis Database dfrA 1461
Escherichia coli Database dhfrIa 1470 Escherichia coli Database
dhfrIb 1475 Escherichia coli Database dhfrV 1480 Proteus mirabilis
Database dhfrVI 1489 Escherichia coli Database dhfrVII 1494
Escherichia coli Database dhfrVIII 1499 Escherichia coli Database
dhfrIX 1504 Escherichia coli Database dhfrXII 1507 Escherichia coli
Database dhfrXIII 1512 Escherichia coli Database dhfrXV 1517
Escherichia coli Database dhfrXVII 1518 Acinetobacter lwoffii This
patent fusA 1519 Acinetobacter lwoffii This patent fusA-tuf spacer
1520 Acinetobacter lwoffii This patent tuf 1521 Haemophilus
influenzae This patent fusA 1522 Haemophilus influenzae This patent
fusA-tuf spacer 1523 Haemophilus influenzae This patent tuf 1524
Proteus mirabilis This patent fusA 1525 Proteus mirabilis This
patent fusA-tuf spacer 1526 Proteus mirabilis This patent fuf 1527
Campylobacter curvus This patent atpD 1530 Escherichia coli
Database ereA 1535 Escherichia coli Database ereB 1540
Staphylococcus haemolyticus Database linA 1545 Enterococcus faecium
Database linB 1548 Streptococcus pyogenes Database mefA 1551
Streptococcus pneumoniae Database mefE 1560 Escherichia coli
Database mphA 1561 Candida albicans This patent tuf (EF-1) 1562
Candida dubliniensis This patent tuf (EF-1) 1563 Candida famata
This patent tuf (EF-1) 1564 Candida glabrata This patent tuf (EF-1)
1565 Candida guilliermondii This patent tuf (EF-1) 1566 Candida
haemulonii This patent tuf (EF-1) 1567 Candida kefyr This patent
tuf (EF-1) 1568 Candida lusitaniae This patent tuf (EF-1) 1569
Candida sphaerica This patent tuf (EF-1) 1570 Candida tropicalis
This patent tuf (EF-1) 1571 Candida viswanathii This patent tuf
(EF-1) 1572 Alcaligenes faecalis subsp. faecalis This patent tuf
1573 Prevotella buccalis This patent tuf 1574 Succinivibrio
dextrinosolvens This patent tuf 1575 Tetragenococcus halophilus
This patent tuf 1576 Campylobacter jejuni subsp. jejuni This patent
atpD 1577 Campylobacter rectus This patent atpD 1578 Enterococcus
casseliflavus This patent fusA 1579 Enterococcus gallinarum This
patent fusA 1580 Streptococcus mitis This patent fusA 1585
Enterococcus faecium Database satG 1590 Cloning vector pFW16
Database tetM 1594 Enterococcus faecium Database vanD 1599
Enterococcus faecalis Database vanE 1600 Campylobacter jejuni
subsp. doylei This patent atpD 1601 Enterococcus sulfurous This
patent atpD 1602 Enterococcus solitarius This patent atpD 1603
Campylobacter sputorum subsp. sputorum This patent atpD 1604
Enterococcus pseudoavium This patent atpD 1607 Klebsiella
omithinolytica This patent gyrA 1608 Klebsiella oxytoca This patent
gyrA 1613 Staphylococcus aureus Database vatB 1618 Staphylococcus
cohnii Database vatC 1623 Staphylococcus aureus Database vga 1628
Staphylococcus aureus Database vgaB 1633 Staphylococcus aureus
Database vgb 1638 Aspergillus fumigatus This patent atpD 1639
Aspergillus fumigatus This patent atpD 1640 Bacillus mycoides This
patent atpD 1641 Bacillus mycoides This patent atpD 1642 Bacillus
mycoides This patent atpD 1643 Bacillus pseudomycoides This patent
atpD 1644 Bacillus pseudomycoides This patent atpD 1645 Budvicia
aquatica This patent atpD 1646 Buttiauxella agrestis This patent
atpD 1647 Candida norvegica This patent atpD 1648 Streptococcus
pneumoniae This patent pbp1a 1649 Campylobacter lari This patent
atpD 1650 Coccidioides immitis This patent atpD 1651 Emmonsia parva
This patent atpD 1652 Erwinia amylovora This patent atpD 1653
Fonsecaea pedrosoi This patent atpD 1654 Fusarium moniliforme This
patent atpD 1655 Klebsiella oxytoca This patent atpD 1656
Microsporum audouinii This patent atpD 1657 Obesumbacterium proteus
This patent atpD 1658 Paracoccidioides brasiliensis This patent
atpD 1659 Plesiomonas shigelloides This patent atpD 1660 Shewanella
putrefaciens This patent atpD 1662 Campylobacter curvus This patent
tuf 1663 Campylobacter rectus This patent tuf 1664 Fonsecaea
pedrosoi This patent tuf 1666 Microsporum audouinii This patent tuf
1667 Piedraia hortai This patent tuf 1668 Escherichia coli Database
tuf 1669 Saksenaea vasiformis This patent tuf 1670 Trichophyton
tonsurans This patent tuf 1671 Enterobacter aerogenes This patent
atpD 1672 Bordetella pertussis Database atpD 1673 Arcanobacterium
haemolyticum This patent tuf 1674 Butyrivibrio fibrisolvens This
patent tuf 1675 Campylobacter jejuni subsp. doylei This patent tuf
1676 Campylobacter lari This patent tuf 1677 Campylobacter sputorum
subsp. sputorum This patent tuf 1678 Campylobacter upsaliensis This
patent tuf 1679 Globicatella sanguis This patent tuf 1680
Lactobacillus acidophilus This patent tuf 1681 Leuconostoc
mesenteroides subsp. dextranicum This patent tuf 1682 Prevotella
buccalis This patent tuf 1683 Ruminococcus bromii This patent tuf
1684 Paracoccidioides brasiliensis This patent atpD 1685 Candida
norvegica This patent tuf (EF-1) 1686 Aspergillus nidulans This
patent tuf 1687 Aspergillus terreus This patent tuf 1688 Candida
norvegica This patent tuf 1689 Candida parapsilosis This patent tuf
1702 Streptococcus gordonii WO98/20157 recA 1703 Streptococcus
mutans WO98/20157 recA 1704 Streptococcus pneumoniae WO98/20157
recA 1705 Streptococcus pyogenes WO98/20157 recA 1706 Streptococcus
salivarius subsp. thermophilus WO98/20157 recA 1707 Escherichia
coli WO98/20157 oxa 1708 Enterococcus faecalis WO98/20157 blaZ 1709
Pseudomonas aeruginosa WO98/20157 aac6'-IIa 1710 Staphylococcus
aureus WO98/20157 ermA 1711 Escherichia coli WO98/20157 ermB 1712
Staphylococcus aureus WO98/20157 ermC 1713 Enterococcus faecalis
WO98/20157 vanB 1714 Campylobacter jejuni subsp. jejuni This patent
recA 1715 Abiotrophia adiacens WO98/20157 tuf 1716 Abiotrophia
defectiva WO98/20157 tuf 1717 Corynebacterium accolens WO98/20157
tuf 1718 Corynebacterium genitalium WO98/20157 tuf 1719
Corynebacterium jeikeium WO98/20157 tuf 1720 Corynebacterium
pseudodiphtheriticum WO98/20157 tuf 1721 Corynebacterium striatum
WO98/20157 tuf 1722 Enterococcus avium WO98/20157 tuf 1723
Gardnerella vaginalis WO98/20157 tuf 1724 Listeria innocua
WO98/20157 tuf 1725 Listeria ivanovii WO98/20157 tuf 1726 Listeria
monocytogenes WO98/20157 tuf 1727 Listeria seeligeri WO98/20157 tuf
1728 Staphylococcus aureus WO98/20157 tuf 1729 Staphylococcus
saprophyticus WO98/20157 tuf 1730 Staphylococcus simulans
WO98/20157 tuf 1731 Streptococcus agalactiae WO98/20157 tuf 1732
Streptococcus pneumoniae WO98/20157 tuf 1733 Streptococcus
salivarius WO98/20157 tuf 1734 Agrobacterium radiobacter WO98/20157
tuf 1735 Bacillus subtilis WO98/20157 tuf 1736 Bacteroides fragilis
WO98/20157 tuf 1737 Borrelia burgdorferi WO98/20157 tuf 1738
Brevibacterium linens WO98/20157 tuf 1739 Chlamydia trachomatis
WO98/20157 tuf 1740 Fibrobacter succinogenes WO98/20157 tuf 1741
Flavobacterium ferrugineum WO98/20157 tuf 1742 Helicobacter pylori
WO98/20157 tuf 1743 Micrococcus luteus WO98/20157 tuf 1744
Mycobacterium tuberculosis WO98/20157 tuf 1745 Mycoplasma
genitalium WO98/20157 tuf 1746 Neisseria gonorrhoeae WO98/20157 tuf
1747 Rickettsia prowazekii WO98/20157 tuf 1748 Salmonella
choleraesuis subsp. choleraesuis WO98/20157 tuf serotype
Typhimurium 1749 Shewanella putrefaciens WO98/20157 tuf 1750
Stigmatella aurantiaca WO98/20157 tuf 1751 Thiomonas cuprina
WO98/20157 tuf 1752 Treponema pallidum WO98/20157 tuf 1753
Ureaplasma urealyticum WO98/20157 tuf 1754 Wolinella succinogenes
WO98/20157 tuf 1755 Burkholderia cepacia WO98/20157 tuf 1756
Bacillus anthracis This patent recA 1757 Bacillus anthracis This
patent recA 1758 Bacillus cereus This patent recA 1759 Bacillus
cereus This patent recA 1760 Bacillus mycoides This patent recA
1761 Bacillus pseudomycoides This patent recA 1762 Bacillus
thuringiensis This patent recA 1763 Bacillus thuringiensis This
patent recA 1764 Klebsiella oxytoca This patent gyrA 1765
Klebsiella pneumoniae subsp. ozaenae This patent gyrA 1766
Klebsiella planticola This patent gyrA 1767 Klebsiella pneumoniae
This patent gyrA 1768 Klebsiella pneumoniae subsp. pneumoniae This
patent gyrA 1769 Klebsiella pneumoniae subsp. pneumoniae This
patent gyrA 1770 Klebsiella pneumoniae subsp. rhinoscleromatis This
patent gyrA 1771 Klebsiella terrigena This patent gyrA 1772
Legionella pneumophila subsp. pneumophila This patent gyrA 1773
Proteus mirabilis This patent gyrA 1774 Providencia rettgeri This
patent gyrA 1775 Proteus vulgaris This patent gyrA 1776 Yersinia
enterocolitica This patent gyrA 1777 Klebsiella oxytoca This patent
parC (grlA) 1778 Klebsiella oxytoca This patent parC (grlA) 1779
Klebsiella pneumoniae subsp. ozaenae This patent parC (grlA) 1780
Klebsiella planticola This patent parC (grlA) 1781 Klebsiella
pneumoniae This patent parC (grlA) 1782 Klebsiella pneumoniae
subsp. pneumoniae This patent parC (grlA) 1783 Klebsiella
pneumoniae subsp. pneumoniae This patent parC (grlA) 1784
Klebsiella pneumoniae subsp. rhinoscleromatis This patent parC
(grlA) 1785 Klebsiella terrigena This patent parC (grlA) 1786
Bacillus cereus This patent fusA 1787 Bacillus cereus This patent
fusA 1788 Bacillus anthracis This patent fusA 1789 Bacillus cereus
This patent fusA 1790 Bacillus anthracis This patent fusA 1791
Bacillus pseudomycoides This patent fusA 1792 Bacillus cereus This
patent fusA 1793 Bacillus anthracis This patent fusA 1794 Bacillus
cereus This patent fusA 1795 Bacillus weihenstephanensis This
patent fusA 1796 Bacillus mycoides This patent fusA 1797 Bacillus
thuringiensis This patent fusA 1798 Bacillus weihenstephanensis
This patent fusA-tuf spacer 1799 Bacillus thuringiensis This patent
fusA-tuf spacer 1800 Bacillus anthracis This patent fusA-tuf spacer
1801 Bacillus pseudomycoides This patent fusA-tuf spacer 1802
Bacillus anthracis This patent fusA-tuf spacer 1803 Bacillus cereus
This patent fusA-tuf spacer 1804 Bacillus cereus This patent
fusA-tuf spacer 1805 Bacillus mycoides This patent fusA-tuf spacer
1806 Bacillus cereus This patent fusA-tuf spacer 1807 Bacillus
cereus This patent fusA-tuf spacer 1808 Bacillus cereus This patent
fusA-tuf spacer 1809 Bacillus anthracis This patent fusA-tuf spacer
1810 Bacillus mycoides This patent tuf 1811 Bacillus thuringiensis
This patent tuf 1812 Bacillus cereus This patent tuf 1813 Bacillus
weihenstephanensis This patent tuf 1814 Bacillus anthracis This
patent tuf 1815 Bacillus cereus This patent tuf 1816 Bacillus
cereus This patent tuf 1817 Bacillus anthracis This patent tuf 1818
Bacillus cereus This patent tuf 1819 Bacillus anthracis This patent
tuf 1820 Bacillus pseudomycoides This patent tuf 1821 Bacillus
cereus This patent tuf 1822 Streptococcus oralis This patent fusA
1823 Budvicia aquatica This patent fusA 1824 Buttiauxella agrestis
This patent fusA 1825 Klebsiella oxytoca This patent fusA 1826
Plesiomonas shigelloides This patent fusA 1827 Shewanella
putrefaciens This patent fusA 1828 Obesumbacterium proteus This
patent fusA
1829 Klebsiella oxytoca This patent fusA-tuf spacer 1830 Budvicia
aquatica This patent fusA-tuf spacer 1831 Plesiomonas shigelloides
This patent fusA-tuf spacer 1832 Obesumbacterium proteus This
patent fusA-tuf spacer 1833 Shewanella putrefaciens This patent
fusA-tuf spacer 1834 Buttiauxella agrestis This patent fusA-tuf
spacer 1835 Campylobacter coli This patent tuf 1836 Campylobacter
fetus subsp. fetus This patent tuf 1837 Campylobacter fetus subsp.
venerealis This patent tuf 1838 Buttiauxella agrestis This patent
tuf 1839 Klebsiella oxytoca This patent tuf 1840 Plesiomonas
shigelloides This patent tuf 1841 Shewanella putrefaciens This
patent tuf 1842 Obesumbacterium proteus This patent tuf 1843
Budvicia aquatica This patent tuf 1844 Abiotrophia adiacens This
patent atpD 1845 Arcanobacterium haemolyticum This patent atpD 1846
Basidiobolus ranarum This patent atpD 1847 Blastomyces dermatitidis
This patent atpD 1848 Blastomyces dermatitidis This patent atpD
1849 Campylobacter coli This patent atpD 1850 Campylobacter fetus
subsp. fetus This patent atpD 1851 Campylobacter fetus subsp.
venerealis This patent atpD 1852 Campylobacter gracilis This patent
atpD 1853 Campylobacter jejuni subsp. jejuni This patent atpD 1854
Enterococcus cecorum This patent atpD 1855 Enterococcus columbae
This patent atpD 1856 Enterococcus dispar This patent atpD 1857
Enterococcus malodoratus This patent atpD 1858 Enterococcus mundtii
This patent atpD 1859 Enterococcus raffinosus This patent atpD 1860
Globicatella sanguis This patent atpD 1861 Lactococcus garvieae
This patent atpD 1862 Lactococcus lactis This patent atpD 1863
Listeria ivanovii This patent atpD 1864 Succinivibrio
dextrinosolvens This patent atpD 1865 Tetragenococcus halophilus
This patent atpD 1866 Campylobacter fetus subsp. fetus This patent
recA 1867 Campylobacter fetus subsp. venerealis This patent recA
1868 Campylobacter jejuni subsp. jejuni This patent recA 1869
Enterococcus avium This patent recA 1870 Enterococcus faecium This
patent recA 1871 Listeria monocytogenes This patent recA 1872
Streptococcus mitis This patent recA 1873 Streptococcus oralis This
patent recA 1874 Aspergillus fumigatus This patent tuf (M) 1875
Aspergillus versicolor This patent tuf (M) 1876 Basidiobolus
ranarum This patent tuf (M) 1877 Campylobacter gracilis This patent
tuf 1878 Campylobacter jejuni subsp. jejuni This patent tuf 1879
Coccidioides immitis This patent tuf (M) 1880 Erwinia amylovora
This patent tuf 1881 Salmonella choleraesuis subsp. choleraesuis
serotype This patent tuf Typhimurium 1899 Klebsiella pneumoniae
Database blaSHV 1900 Klebsiella pneumoniae Database blaSHV 1901
Escherichia coli Database blaSHV 1902 Klebsiella pneumoniae
Database blaSHV 1903 Klebsiella pneumoniae Database blaSHV 1904
Escherichia coli Database blaSHV 1905 Pseudomonas aeruginosa
Database blaSHV 1927 Neisseria meningitidis Database blaTEM 1928
Escherichia coli Database blaTEM 1929 Klebsiella oxytoca Database
blaTEM 1930 Escherichia coli Database blaTEM 1931 Escherichia coli
Database blaTEM 1932 Escherichia coli Database blaTEM 1933
Escherichia coli Database blaTEM 1954 Klebsiella pneumoniae subsp.
pneumoniae Database gyrA 1956 Candida inconspicua This patent tuf
(M) 1957 Candida utilis This patent tuf (M) 1958 Candida
zeylanoides This patent tuf (M) 1959 Candida catenulata This patent
tuf (M) 1960 Candida krusei This patent tuf (M) 1965 Plasmid pGS05
Database sulII 1970 Transposon Tn10 Database tetB 1985 Cryptococcus
neoformans Database tuf (EF-1) 1986 Cryptococcus neoformans
Database tuf (EF-1) 1987 Saccharomyces cerevisiae Database tuf
(EF-1) 1988 Saccharomyces cerevisiae Database tuf (EF-1) 1989
Eremothecium gossypii Database tuf (EF-1) 1990 Eremothecium
gossypii Database tuf (EF-1) 1991 Aspergillus oryzae Database tuf
(EF-1) 1992 Aureobasidium pullulans Database tuf (EF-1) 1993
Histoplasma capsulatum Database tuf (EF-1) 1994 Neurospora crassa
Database tuf (EF-1) 1995 Podospora anserina Database tuf (EF-1)
1996 Podospora curvicolla Database tuf (EF-1) 1997 Sordaria
macrospora Database tuf (EF-1) 1998 Trichoderma reesei Database tuf
(EF-1) 2004 Candida albicans Database tuf (M) 2005
Schizosaccharomyces pombe Database tuf (M) 2010 Klebsiella
pneumoniae Database blaTEM 2011 Klebsiella pneumoniae Database
blaTEM 2013 Kluyvera ascorbata This patent gyrA 2014 Kluyvera
georgiana This patent gyrA 2047 Streptococcus pneumoniae Database
pbp1A 2048 Streptococcus pneumoniae Database pbp1A 2049
Streptococcus pneumoniae Database pbp1A 2050 Streptococcus
pneumoniae Database pbp1A 2051 Streptococcus pneumoniae Database
pbp1A 2052 Streptococcus pneumoniae Database pbp1A 2053
Streptococcus pneumoniae Database pbp1A 2054 Streptococcus
pneumoniae Database gyrA 2055 Streptococcus pneumoniae Database
parC 2056 Streptococcus pneumoniae This patent pbp1A 2057
Streptococcus pneumoniae This patent pbp1A 2058 Streptococcus
pneumoniae This patent pbp1A 2059 Streptococcus pneumoniae This
patent pbp1A 2060 Streptococcus pneumoniae This patent pbp1A 2061
Streptococcus pneumoniae This patent pbp1A 2062 Streptococcus
pneumoniae This patent pbp1A 2063 Streptococcus pneumoniae This
patent pbp1A 2064 Streptococcus pneumoniae This patent pbp1A 2072
Mycobacterium tuberculosis Database rpoB 2097 Mycoplasma pneumoniae
Database tuf 2101 Mycobacterium tuberculosis Database inhA 2105
Mycobacterium tuberculosis Database embB 2129 Clostridium difficile
Database cdtA 2130 Clostridium difficile Database cdtB 2137
Pseudomonas putida Genome project tuf 2138 Pseudomonas aeruginosa
Genome project tuf 2139 Campylobacter jejuni Database atpD 2140
Streptococcus pneumoniae Database pbp1a 2144 Staphylococcus aureus
Database mupA 2147 Escherichia coli Database catI 2150 Escherichia
coli Database catII 2153 Shigella flexneri Database catIII 2156
Clostridium perfringens Database catP 2159 Staphylococcus aureus
Database cat 2162 Staphylococcus aureus Database cat 2165
Salmonella typhimurium Database ppflo-like 2183 Alcaligenes
faecalis subsp. faecalis This patent tuf 2184 Campylobacter coli
This patent fusA 2185 Succinivibrio dextrinosolvens This patent tuf
2186 Tetragenococcus halophilus This patent tuf 2187 Campylobacter
jejuni subsp. jejuni This patent fusA 2188 Campylobacter jejuni
subsp. jejuni This patent fusA 2189 Leishmania guyanensis This
patent atpD 2190 Trypanosoma brucei brucei This patent atpD 2191
Aspergillus nidulans This patent atpD 2192 Leishmania panamensis
This patent atpD 2193 Aspergillus nidulans This patent tuf (M) 2194
Aureobasidium pullulans This patent tuf (M) 2195 Emmonsia parva
This patent tuf (M) 2196 Exserohilum rostratum This patent tuf (M)
2197 Fusarium moniliforme This patent tuf (M) 2198 Fusarium solani
This patent tuf (M) 2199 Histoplasma capsulatum This patent tuf (M)
2200 Kocuria kristinae This patent tuf 2201 Vibrio mimicus This
patent tuf 2202 Citrobacter freundii This patent recA 2203
Clostridium botulinum This patent recA 2204 Francisella tularensis
This patent recA 2205 Peptostreptococcus anaerobius This patent
recA 2206 Peptostreptococcus asaccharolyticus This patent recA 2207
Providencia stuartii This patent recA 2208 Salmonella choleraesuis
subsp. choleraesuis This patent recA serotype Paratyphi A 2209
Salmonella choleraesuis subsp. choleraesuis This patent recA
serotype Typhimurium 2210 Staphylococcus saprophyticus This patent
recA 2211 Yersinia pseudotuberculosis This patent recA 2212
Zoogloea ramigera This patent recA 2214 Abiotrophia adiacens This
patent fusA 2215 Acinetobacter baumannii This patent fusA 2216
Actinomyces meyeri This patent fusA 2217 Clostridium difficile This
patent fusA 2218 Corynebacterium diphtheriae This patent fusA 2219
Enterobacter cloacae This patent fusA 2220 Klebsiella pneumoniae
subsp. pneumoniae This patent fusA 2221 Listeria monocytogenes This
patent fusA 2222 Mycobacterium avium This patent fusA 2223
Mycobacterium gordonae This patent fusA 2224 Mycobacterium kansasii
This patent fusA 2225 Mycobacterium terrae This patent fusA 2226
Neisseria polysaccharea This patent fusA 2227 Staphylococcus
epidermidis This patent fusA 2228 Staphylococcus haemolyticus This
patent fusA 2229 Succinivibrio dextrinosolvens This patent fusA
2230 Tetragenococcus halophilus This patent fusA 2231 Veillonella
parvula This patent fusA 2232 Yersinia pseudotuberculosis This
patent fusA 2233 Zoogloea ramigera This patent fusA 2234 Aeromonas
hydrophila This patent fusA 2235 Abiotrophia adiacens This patent
fusA-tuf spacer 2236 Acinetobacter baumannii This patent fusA-tuf
spacer 2237 Actinomyces meyeri This patent fusA-tuf spacer 2238
Clostridium difficile This patent fusA-tuf spacer 2239
Corynebacterium diphtheriae This patent fusA-tuf spacer 2240
Enterobacter cloacae This patent fusA-tuf spacer 2241 Klebsiella
pneumoniae subsp. pneumoniae This patent fusA-tuf spacer 2242
Listeria monocytogenes This patent fusA-tuf spacer 2243
Mycobacterium avium This patent fusA-tuf spacer 2244 Mycobacterium
gordonae This patent fusA-tuf spacer 2245 Mycobacterium kansasii
This patent fusA-tuf spacer 2246 Mycobacterium terrae This patent
fusA-tuf spacer 2247 Neisseria polysaccharea This patent fusA-tuf
spacer 2248 Staphylococcus epidermidis This patent fusA-tuf spacer
2249 Staphylococcus haemolyticus This patent fusA-tuf spacer 2255
Abiotrophia adiacens This patent tuf 2256 Acinetobacter baumannii
This patent tuf 2257 Actinomyces meyeri This patent tuf 2258
Clostridium difficile This patent tuf 2259 Corynebacterium
diphtheriae This patent tuf 2260 Enterobacter cloacae This patent
tuf 2261 Klebsiella pneumoniae subsp. pneumoniae This patent tuf
2262 Listeria monocytogenes This patent tuf 2263 Mycobacterium
avium This patent tuf 2264 Mycobacterium gordonae This patent tuf
2265 Mycobacterium kansasii This patent tuf 2266 Mycobacterium
terrae This patent tuf 2267 Neisseria polysaccharea This patent tuf
2268 Staphylococcus epidermidis This patent tuf 2269 Staphylococcus
haemolyticus This patent tuf 2270 Aeromonas hydrophila This patent
tuf 2271 Bilophila wadsworthia This patent tuf 2272 Brevundimonas
diminuta This patent tuf 2273 Streptococcus mitis This patent pbp1a
2274 Streptococcus mitis This patent pbp1a 2275 Streptococcus mitis
This patent pbp1a 2276 Streptococcus oralis This patent pbp1a 2277
Escherichia coli This patent gyrA 2278 Escherichia coli This patent
gyrA 2279 Escherichia coli This patent gyrA 2280 Escherichia coli
This patent gyrA 2288 Enterococcus faecium Database ddl 2293
Enterococcus faecium Database vanA 2296 Enterococcus faecalis
Database vanB *tuf indicates tuf sequences, tuf (C) indicates tuf
sequences divergent from main (usually A and B) copies of the
elongation factor-Tu, tuf (EF-1) indicates tuf sequences of the
eukaryotic type (elongation factor 1.alpha.), tuf (M) indicates tuf
sequences from organellar (mostly mitochondrial) origin. fusA
indicates fusA sequences; fusA-tuf spacer indicates the intergenic
region between fusA and tuf. atpD indicates atpD sequences of the
F-type, atpD (V) indicates atpD sequences of the V-type. recA
indicates recA sequences, recA(Rad51) indicates rad51 sequences or
homologs and recA(Dmc1) indicates dmc1 sequences or homologs.
TABLE-US-00009 TABLE 8 Bacterial species used to test the
specificity of the Streptococcus agalactiae-specific amplification
primers derived from tuf sequences. Strain Reference number Strain
Reference number Streptococcus acidominimus ATCC 51726 Bacteroides
caccae ATCC 43185 Streptococcus agalactiae ATCC 12403 Bacteroides
vulgatus ATCC 8482 Streptococcus agalactiae ATCC 12973 Bacteroides
fragilis ATCC 25285 Streptococcus agalactiae ATCC 13813 Candida
albicans ATCC 11006 Streptococcus agalactiae ATCC 27591 Clostridium
innoculum ATCC 14501 Streptococcus agalactiae CDCs 1073 Clostridium
ramosum ATCC 25582 Streptococcus anginosus ATCC 27335 Lactobacillus
casei subsp. casei ATCC 393 Streptococcus anginosus ATCC 33397
Clostridium septicum ATCC 12464 Streptococcus bovis ATCC 33317
Corynebacterium cervicis NCTC 10604 Streptococcus anginosus ATCC
27823 Corynebacterium genitalium ATCC 33031 Streptococcus cricetus
ATCC 19642 Corynebacterium urealyticum ATCC 43042 Streptococcus
cristatus ATCC 51100 Enterococcus faecalis ATCC 29212 Streptococcus
downei ATCC 33748 Enterococcus faecium ATCC 19434 Streptococcus
dysgalactiae ATCC 43078 Eubacterium lentum ATCC 43055 Streptococcus
equi subsp. equi ATCC 9528 Eubacterium nodutum ATCC 33099
Streptococcus ferus ATCC 33477 Gardnerella vaginalis ATCC 14018
Streptococcus gordonii ATCC 10558 Lactobacillus acidophilus ATCC
4356 Streptococcus macacae ATCC 35911 Lactobacillus crispatus ATCC
33820 Streptococcus mitis ATCC 49456 Lactobacillus gasseri ATCC
33323 Streptococcus mutans ATCC 25175 Lactobacillus johnsonii ATCC
33200 Streptococcus oralis ATCC 35037 Lactococcus lactis subsp.
lactis ATCC 19435 Streptococcus parasanguinis ATCC 15912
Lactococcus lactis subsp. lactis ATCC 11454 Streptococcus
parauberis DSM 6631 Listeria innocua ATCC 33090 Streptococcus
pneumoniae ATCC 27336 Micrococcus luteus ATCC 9341 Streptococcus
pyogenes ATCC 19615 Escherichia coli ATCC 25922 Streptococcus ratti
ATCC 19645 Micrococcus lylae ATCC 27566 Streptococcus salivarius
ATCC 7073 Porphyromonas asaccharolytica ATCC 25260 Streptococcus
sanguinis ATCC 10556 Prevotella corporis ATCC 33547 Streptococcus
sobrinus ATCC 27352 Prevotella melanogenica ATCC 25845
Streptococcus suis ATCC 43765 Staphylococcus aureus ATCC 13301
Streptococcus uberis ATCC 19436 Staphylococcus epidermidis ATCC
14990 Streptococcus vestubularis ATCC 49124 Staphylococcus
saprophyticus ATCC 15305
TABLE-US-00010 TABLE 9 Bacterial species used to test the
specificity of the Streptococcus agalactiae-specific amplification
primers derived from atpD sequences. Strain Reference number
Streptococcus acidominimus ATCC 51726 Streptococcus agalactiae ATCC
12400 Streptococcus agalactiae ATCC 12403 Streptococcus agalactiae
ATCC 12973 Streptococcus agalactiae ATCC 13813 Streptococcus
agalactiae ATCC 27591 Streptococcus agalactiae CDCs-1073
Streptococcus anginosus ATCC 27335 Streptococcus anginosus ATCC
27823 Streptococcus bovis ATCC 33317 Streptococcus cricetus ATCC
19642 Streptococcus cristatus ATCC 51100 Streptococcus downei ATCC
33748 Streptococcus dysgalactiae ATCC 43078 Streptococcus equi
subsp. equi ATCC 9528 Streptococcus ferus ATCC 33477 Streptococcus
gordonii ATCC 10558 Streptococcus macacae ATCC 35911 Streptococcus
mitis ATCC 49456 Streptococcus mutans ATCC 25175 Streptococcus
oralis ATCC 35037 Streptococcus parasanguinis ATCC 15912
Streptococcus parauberis DSM 6631 Streptococcus pneumoniae ATCC
27336 Streptococcus pyogenes ATCC 19615 Streptococcus ratti ATCC
19645 Streptococcus salivarius ATCC 7073 Streptococcus sanguinis
ATCC 10556 Streptococcus sobrinus ATCC 27352 Streptococcus suis
ATCC 43765 Streptococcus uberis ATCC 19436 Streptococcus
vestibularis ATCC 49124
TABLE-US-00011 TABLE 10 Bacterial species used to test the
specificity of the Enterococcus-specific amplification primers
derived from tuf sequences. Strain Reference number Gram-positive
species (n = 74) Abiotrophia adiacens ATCC 49176 Abiotrophia
defectiva ATCC 49175 Bacillus cereus ATCC 14579 Bacillus subtilis
ATCC 27370 Bifidobacterium adolescentis ATCC 27534 Bifidobacterium
breve ATCC 15700 Bifidobacterium dentium ATCC 27534 Bifidobacterium
longum ATCC 15707 Clostridium perfringens ATCC 3124 Clostridium
septicum ATCC 12464 Corynebacterium aquaticus ATCC 14665
Corynebacterium ATCC 10700 pseudodiphtheriticum Enterococcus avium
ATCC 14025 Enterococcus casseliflavus ATCC 25788 Enterococcus
cecorum ATCC 43199 Enterococcus columbae ATCC 51263 Enterococcus
dispar ATCC 51266 Enterococcus durans ATCC 19432 Enterococcus
faecalis ATCC 29212 Enterococcus faecium ATCC 19434 Enterococcus
flavescens ATCC 49996 Enterococcus gallinarum ATCC 49573
Enterococcus hirae ATCC 8044 Enterococcus malodoratus ATCC 43197
Enterococcus mundtii ATCC 43186 Enterococcus pseudoavium ATCC 49372
Enterococcus raffinosus ATCC 49427 Enterococcus saccharolyticus
ATCC 43076 Enterococcus solitarius ATCC 49428 Enterococcus
sulfureus ATCC 49903 Eubacterium lentum ATCC 49903 Gemella
haemolysans ATCC 10379 Gemella morbillorum ATCC 27842 Lactobacillus
acidophilus ATCC 4356 Leuconostoc mesenteroides ATCC 19225 Listeria
grayi ATCC 19120 Listeria grayi ATCC 19123 Listeria innocua ATCC
33090 Listeria ivanovii ATCC 19119 Listeria monocytogenes ATCC
15313 Listeria seeligeri ATCC 35967 Micrococcus luteus ATCC 9341
Pediococcus acidilacti ATCC 33314 Pediococcus pentosaceus ATCC
33316 Peptococcus niger ATCC 27731 Peptostreptococcus anaerobius
ATCC 27337 Peptostreptococcus indolicus ATCC 29247
Peptostreptococcus micros ATCC 33270 Propionibacterium acnes ATCC
6919 Staphylococcus aureus ATCC 43300 Staphylococcus capitis ATCC
27840 Staphylococcus epidermidis ATCC 14990 Staphylococcus
haemolyticus ATCC 29970 Staphylococcus hominis ATCC 27844
Staphylococcus lugdunensis ATCC 43809 Staphylococcus saprophyticus
ATCC 15305 Staphylococcus simulans ATCC 27848 Staphylococcus
warneri ATCC 27836 Streptococcus agalactiae ATCC 13813
Streptococcus anginosus ATCC 33397 Streptococcus bovis ATCC 33317
Streptococcus constellatus ATCC 27823 Streptococcus cristatus ATCC
51100 Streptococcus intermedius ATCC 27335 Streptococcus mitis ATCC
49456 Streptococcus mitis ATCC 3639 Streptococcus mutans ATCC 27175
Streptococcus parasanguinis ATCC 15912 Streptococcus pneumoniae
ATCC 27736 Streptococcus pneumoniae ATCC 6303 Streptococcus
pyogenes ATCC 19615 Streptococcus salivarius ATCC 7073
Streptococcus sanguinis ATCC 10556 Streptococcus suis ATCC 43765
Gram-negative species (n = 39) Acidominococcus fermentans ATCC 2508
Acinetobacter baumannii ATCC 19606 Alcaligenes faecalis ATCC 8750
Anaerobiospirillum ATCC 29305 succiniproducens Anaerorhabdus
furcosus ATCC 25662 Bacteroides distasonis ATCC 8503 Bacteroides
thetaiotaomicron ATCC 29741 Bacteroides vulgatus ATCC 8482
Bordetella pertussis LSPQ 3702 Bulkholderia cepacia LSPQ 2217
Butyvibrio fibrinosolvens ATCC 19171 Cardiobacterium hominis ATCC
15826 Citrobacter freundii ATCC 8090 Desulfovibrio vulgaris ATCC
29579 Edwardsiellae tarda ATCC 15947 Enterobacter cloacae ATCC
13047 Escherichia coli ATCC 25922 Fusobacterium russii ATCC 25533
Haemophilus influenzae ATCC 9007 Hafnia alvei ATCC 13337 Klebsiella
oxytoca ATCC 13182 Meganomonas hypermegas ATCC 25560 Mitsukoella
multiacidus ATCC 27723 Moraxella catarrhalis ATCC 43628 Morganella
morganii ATCC 25830 Neisseria meningitidis ATCC 13077 Pasteurella
aerogenes ATCC 27883 Proteus vulgaris ATCC 13315 Providencia
alcalifaciens ATCC 9886 Providencia rettgeri ATCC 9250 Pseudomonas
aeruginosa ATCC 27853 Salmonella typhimurium ATCC 14028 Serratia
marcescens ATCC 13880 Shigella flexneri ATCC 12022 Shigella sonnei
ATCC 29930 Succinivibrio dextrinosolvens ATCC 19716 Tissierella
praeacuta ATCC 25539 Veillonella parvuala ATCC 10790 Yersinia
enterocolitica ATCC 9610
TABLE-US-00012 TABLE 11 Microbial species for which tuf and/or atpD
and/or recA sequences are available in public databases. Species
Strain Accession number Coding gene* tuf sequences Bacteria
Actinobacillus actinomycetemcomitans HK1651 Genome project.sup.2
tuf Actinobacillus actinomycetemcomitans HK1651 Genome
project.sup.2 tuf (EF-G) Agrobacterium tumefaciens X99673 tuf
Agrobacterium tumefaciens X99673 tuf (EF-G) Agrobacterium
tumefaciens X99674 tuf Anacystis nidulans PCC 6301 X17442 tuf
Aquifex aeolicus VF5 AE000669 tuf Aquifex aeolicus VF5 AE000669 tuf
(EF-G) Aquifex pyrophilus Genome project.sup.2 tuf (EF-G) Aquifex
pyrophilus Y15787 tuf Bacillus anthracis Ames Genome project.sup.2
tuf Bacillus anthracis Ames Genome project.sup.2 tuf (EF-G)
Bacillus halodurans C-125 AB017508 tuf Bacillus halodurans C-125
AB017508 tuf (EF-G) Bacillus stearothermophilus CCM 2184 AJ000260
tuf Bacillus subtilis 168 D64127 tuf Bacillus subtilis 168 D64127
tuf (EF-G) Bacillus subtilis DSM 10 Z99104 tuf Bacillus subtilis
DSM 10 Z99104 tuf (EF-G) Bacteroides forsythus ATCC 43037 AB035466
tuf Bacteroides fragilis DSM 1151 --.sup.1 tuf Bordetella
bronchiseptica RB50 Genome project.sup.2 tuf Bordetella pertussis
Tohama 1 Genome project.sup.2 tuf Bordetella pertussis Tohama 1
Genome project.sup.2 tuf (EF-G) Borrelia burdorgferi B31 U78193 tuf
Borrelia burgdorferi AE001155 tuf (EF-G) Brevibacterium linens DSM
20425 X76863 tuf Buchnera aphidicola Ap Y12307 tuf Burkholderia
pseudomallei K96243 Genome project.sup.2 tuf (EF-G) Campylobacter
jejuni NCTC 11168 Y17167 tuf Campylobacter jejuni NCTC 11168
CJ11168X2 tuf (EF-G) Chlamydia pneumoniae CWL029 AE001592 tuf
Chlamydia pneumoniae CWL029 AE001639 tuf (EF-G) Chlamydia
trachomatis M74221 tuf Chlamydia trachomatis D/UW-3/CX AE001317 tuf
(EF-G) Chlamydia trachomatis D/UW-3/CX AE001305 tuf Chlamydia
trachomatis F/IC-Cal-13 L22216 tuf Chlorobium vibrioforme DSM 263
X77033 tuf Chloroflexus aurantiacus DSM 636 X76865 tuf Clostridium
acetobutylicum ATCC 824 Genome project.sup.2 tuf Clostridium
difficile 630 Genome project.sup.2 tuf Clostridium difficile 630
Genome project.sup.2 tuf (EF-G) Corynebacterium diphtheriae NCTC
13129 Genome project.sup.2 tuf Corynebacterium diphtheriae NCTC
13129 Genome project.sup.2 tuf (EF-G) Corynebacterium glutamicum
ASO 19 X77034 tuf Corynebacterium glutamicum MJ-233 E09634 tuf
Coxiella burnetii Nine Mile phase I AF136604 tuf Cytophaga lytica
DSM 2039 X77035 tuf Deinococcus radiodurans R1 AE001891 tuf (EF-G)
Deinococcus radiodurans R1 AE180092 tuf Deinococcus radiodurans R1
AE002041 tuf Deinonema sp. --.sup.1 tuf Eikenella corrodens ATCC
23834 Z12610 tuf Eikenella corrodens ATCC 23834 Z12610 tuf (EF-G)
Enterococcus faecalis Genome project.sup.2 tuf (EF-G) Escherichia
coli J01690 tuf Escherichia coli J01717 tuf Escherichia coli X00415
tuf (EF-G) Escherichia coli X57091 tuf Escherichia coli K-12 MG1655
U00006 tuf Escherichia coli K-12 MG1655 U00096 tuf Escherichia coli
K-12 MG1655 AE000410 tuf (EF-G) Fervidobacterium islandicum DSM
5733 Y15788 tuf Fibrobacter succinogenes S85 X76866 tuf
Flavobacterium ferrigeneum DSM 13524 X76867 tuf Flexistipes
sinusarabici X59461 tuf Gloeobacter violaceus PCC 7421 U09433 tuf
Gloeothece sp. PCC 6501 U09434 tuf Haemophilus
actinomycetemcomitans HK1651 Genome project.sup.2 tuf Haemophilus
ducreyi 35000 AF087414 tuf (EF-G) Haemophilus influenzae Rd U32739
tuf Haemophilus influenzae Rd U32746 tuf Haemophilus influenzae Rd
U32739 tuf (EF-G) Helicobacter pylori 26695 AE000511 tuf
Helicobacter pylori J99 AE001539 tuf (EF-G) Helicobacter pylori J99
AE001541 tuf Herpetosiphon aurantiacus Hpga1 X76868 tuf Klebsiella
pneumoniae M6H 78578 Genome project.sup.2 tuf Klebsiella pneumoniae
M6H 78578 Genome project.sup.2 tuf (EF-G) Lactobacillus paracasei
E13922 tuf Legionella pneumophila Philadelphia-1 Genome
project.sup.2 tuf Leptospira interrogans AF115283 tuf Leptospira
interrogans AF115283 tuf (EF-G) Micrococcus luteus IFO 3333 M17788
tuf (EF-G) Micrococcus luteus IFO 3333 M17788 tuf Moraxella sp. TAC
II 25 AJ249258 tuf Mycobacterium avium 104 Genome project.sup.2 tuf
Mycobacterium avium 104 Genome project.sup.2 tuf (EF-G)
Mycobacterium bovis AF2122/97 Genome project.sup.2 tuf
Mycobacterium bovis AF2122/97 Genome project.sup.2 tuf (EF-G)
Mycobacterium leprae L13276 tuf Mycobacterium leprae Z14314 tuf
Mycobacterium leprae Z14314 tuf (EF-G) Mycobacterium leprae Thai 53
D13869 tuf Mycobacterium tuberculosis Erdmann S40925 tuf
Mycobacterium tuberculosis H37Rv AL021943 tuf (EF-G) Mycobacterium
tuberculosis H37Rv Z84395 tuf Mycobacterium tuberculosis y42
AD000005 tuf Mycobacterium tuberculosis CSU#93 Genome project.sup.2
tuf Mycobacterium tuberculosis CSU#93 Genome project.sup.2 tuf
(EF-G) Mycoplasma capricolum PG-31 X16462 tuf Mycoplasma genitalium
G37 U39732 tuf Mycoplasma genitalium G37 U39689 tuf (EF-G)
Mycoplasma hominis X57136 tuf Mycoplasma hominis PG21 M57675 tuf
Mycoplasma pneumoniae M129 AE000019 tuf Mycoplasma pneumoniae M129
AE000058 tuf (EF-G) Neisseria gonorrhoeae MS11 L36380 tuf Neisseria
gonorrhoeae MS11 L36380 tuf (EF-G) Neisseria meningitidis Z2491
Genome project.sup.2 tuf (EF-G) Neisseria meningitidis Z2491 Genome
project.sup.2 tuf Pasteurella multocida Pm70 Genome project.sup.2
tuf Peptococcus niger DSM 20745 X76869 tuf Phormidium ectocarpi PCC
7375 U09443 tuf Planobispora rosea ATCC 53773 U67308 tuf
Planobispora rosea ATCC 53733 X98830 tuf Planobispora rosea ATCC
53733 X98830 tuf (EF-G) Plectonema boryanum PCC 73110 U09444 tuf
Porphyromonas gingivalis W83 Genome project.sup.2 tuf Porphyromonas
gingivalis W83 Genome project.sup.2 tuf (EF-G) Porphyromonas
gingivalis FDC 381 AB035461 tuf Porphyromonas gingivalis W83
AB035462 tuf Porphyromonas gingivalis SUNY 1021 AB035463 tuf
Porphyromonas gingivalis A7A1-28 AB035464 tuf Porphyromonas
gingivalis ATCC 33277 AB035465 tuf Porphyromonas gingivalis ATCC
33277 AB035471 tuf (EF-G) Prochlorothrix hollandica U09445 tuf
Pseudomonas aeruginosa PAO-1 Genome project.sup.2 tuf Pseudomonas
putida Genome project.sup.2 tuf Rickettsia prowazekii Madrid E
AJ235272 tuf Rickettsia prowazekii Madrid E AJ235270 tuf (EF-G)
Rickettsia prowazekii Madrid E Z54171 tuf (EF-G) Salmonella
choleraesuis subsp. X64591 tuf (EF-G) choleraesuis serotype
Typhimurium Salmonella choleraesuis subsp. LT2 trpE91 X55116 tuf
choleraesuis serotype Typhimurium Salmonella choleraesuis subsp.
LT2 trpE91 X55117 tuf choleraesuis serotype Typhimurium Serpulina
hyodysenteriae B204 U51635 tuf Serratia marcescens AF058451 tuf
Shewanella putrefaciens DSM 50426 --.sup.1 tuf Shewanella
putrefaciens MR-1 Genome project.sup.2 tuf Spirochaeta aurantia DSM
1902 X76874 tuf Staphylococcus aureus AJ237696 tuf (EF-G)
Staphylococcus aureus EMRSA-16 Genome project.sup.2 tuf
Staphylococcus aureus NCTC 8325 Genome project.sup.2 tuf
Staphylococcus aureus COL Genome project.sup.2 tuf Staphylococcus
aureus EMRSA-16 Genome project.sup.2 tuf (EF-G) Stigmatella
aurantiaca DW4 X82820 tuf Stigmatella aurantiaca Sg a1 X76870 tuf
Streptococcus mutans GS-5 Kuramitsu U75481 tuf Streptococcus mutans
UAB159 Genome project.sup.2 tuf Streptococcus oralis NTCC 11427
P331701 tuf Streptococcus pyogenes Genome project.sup.2 tuf (EF-G)
Streptococcus pyogenes M1-GAS Genome project.sup.2 tuf Streptomyces
aureofaciens ATCC 10762 AF007125 tuf Streptomyces cinnamoneus Tue89
X98831 tuf Streptomyces coelicolor A3(2) AL031013 tuf (EF-G)
Streptomyces coelicolor A3(2) X77039 tuf (EF-G) Streptomyces
coelicolor M145 X77039 tuf Streptomyces collinus BSM 40733 S79408
tuf Streptomyces netropsis Tu1063 AF153618 tuf Streptomyces
ramocissimus X67057 tuf Streptomyces ramocissimus X67058 tuf
Streptomyces ramocissimus X67057 tuf (EF-G) Synechococcus sp. PCC
6301 X17442 tuf (EF-G) Synechococcus sp. PCC 6301 X17442 tuf
Synechocystis sp. PCC 6803 D90913 tuf (EF-G) Synechocystis sp. PCC
6803 D90913 tuf Synechocystis sp. PCC 6803 X65159 tuf (EF-G)
Taxeobacter occealus Myx 2105 X77036 tuf Thermotoga maritima Genome
project.sup.2 tuf (EF-G) Thermotoga maritima M27479 tuf Thermus
aquaticus EP 00276 X66322 tuf Thermus thermophilus HB8 X16278 tuf
(EF-G) Thermus thermophilus HB8 X05977 tuf Thermus thermophilus HB8
X06657 tuf Thiomonas cuprina DSM 5495 U78300 tuf Thiomonas cuprina
DSM 5495 U78300 tuf (EF-G) Thiomonas cuprina Hoe5 X76871 tuf
Treponema denticola Genome project.sup.2 tuf Treponema denticola
Genome project.sup.2 tuf (EF-G) Treponema pallidum AE001202 tuf
Treponema pallidum AE001222 tuf (EF-G) Treponema pallidum AE001248
tuf (EF-G) Ureaplasma urealyticum ATCC 33697 Z34275 tuf Ureaplasma
urealyticum serovar 3 biovar 1 AE002151 tuf Ureaplasma urealyticum
serovar 3 biovar 1 AE002151 tuf (EF-G) Vibrio cholerae N16961
Genome project.sup.2 tuf Wolinella succinogenes DSM 1740 X76872 tuf
Yersinia pestis CO-92 Genome project.sup.2 tuf Yersinia pestis
CO-92 Genome project.sup.2 tuf (EF-G) Archaebacteria Archaeoglobus
fulgidus Genome project.sup.2 tuf (EF-G) Halobacterium marismortui
X16677 tuf Methanobacterium thermoautrophicum delta H AE000877 tuf
Methanococcus jannaschii ATCC 43067 U67486 tuf Methanococcus
vannielii X05698 tuf Pyrococcus abyssi Orsay AJ248285 tuf
Thermoplasma acidophilum DSM 1728 X53866 tuf Fungi Absidia glauca
CBS 101.48 X54730 tuf (EF-1) Arxula adeninivorans Ls3 Z47379 tuf
(EF-1) Aspergillus oryzae KBN616 AB007770 tuf (EF-1) Aureobasidium
pullulans R106 U19723 tuf (EF-1) Candida albicans SC5314 Genome
project.sup.2 tuf (M) Candida albicans SC5314 M29934 tuf (EF-1)
Candida albicans SC5314 M29935 tuf (EF-1) Cryptococcus neoformans
B3501 U81803 tuf (EF-1) Cryptococcus neoformans M1-106 U81804 tuf
(EF-1) Eremothecium gossypii ATCC 10895 X73978 tuf (EF-1)
Eremothecium gossypii A29820 tuf (EF-1) Fusarium oxysporum NRRL
26037 AF008498 tuf (EF-1) Histoplasma capsulatum 186AS U14100 tuf
(EF-1) Podospora anserina X74799 tuf (EF-1) Podospora curvicolla
VLV X96614 tuf (EF-1) Prototheca wickerhamii 263-11 AJ245645 tuf
(EF-1) Puccinia graminis race 32 X73529 tuf (EF-1) Reclinomonas
americana ATCC 50394 AF007261 tuf (M) Rhizomucor racemosus ATCC
1216B X17475 tuf (EF-1) Rhizomucor racemosus ATCC 1216B J02605 tuf
(EF-1) Rhizomucor racemosus ATCC 1216B X17476 tuf (EF-1)
Rhodotorula mucilaginosa AF016239 tuf (EF-1) Saccharomyces
cerevisiae K00428 tuf (M) Saccharomyces cerevisiae M59369 tuf
(EF-G) Saccharomyces cerevisiae X00779 tuf (EF-1) Saccharomyces
cerevisiae X01638 tuf (EF-1) Saccharomyces cerevisiae M10992 tuf
(EF-1) Saccharomyces cerevisiae Alpha S288 X78993 tuf (EF-1)
Saccharomyces cerevisiae M15666 tuf (EF-1) Saccharomyces cerevisiae
Z35987 tuf (EF-1) Saccharomyces cerevisiae S288C (AB972) U51033 tuf
(EF-1) Schizophyllum commune 1-40 X94913 tuf (EF-1)
Schizosaccharomyces pombe 972h- AL021816 tuf (EF-1)
Schizosaccharomyces pombe 972h- AL021813 tuf (EF-1)
Schizosaccharomyces pombe 972h- D82571 tuf (EF-1)
Schizosaccharomyces pombe U42189 tuf (EF-1) Schizosaccharomyces
pombe PR745 D89112 tuf (EF-1) Sordaria macrospora OOO X96615 tuf
(EF-1) Trichoderma reesei QM9414 Z23012 tuf (EF-1) Yarrowia
lipolytica AF054510 tuf (EF-1)
Parasites Blastocystis hominis HE87-1 D64080 tuf (EF-1)
Cryptosporidium parvum U69697 tuf (EF-1) Eimeria tenella LS18
AI755521 tuf (EF-1) Entamoeba histolytica HM1:IMSS X83565 tuf
(EF-1) Entamoeba histolytica NIH 200 M92073 tuf (EF-1) Giardia
lamblia D14342 tuf (EF-1) Kentrophoros sp. AF056101 tuf (EF-1)
Leishmania amazonensis IFLA/BR/67/PH8 M92653 tuf (EF-1) Leishmania
braziliensis U72244 tuf (EF-1) Onchocerca volvulus M64333 tuf
(EF-1) Porphyra purpurea Avonport U08844 tuf (EF-1) Plasmodium
berghei ANKA AJ224150 tuf (EF-1) Plasmodium falciparum K1 X60488
tuf (EF-1) Plasmodium knowlesi line H AJ224153 tuf (EF-1)
Toxoplasma gondii RH Y11431 tuf (EF-1) Trichomonas tenax ATCC 30207
D78479 tuf (EF-1) Trypanosoma brucei LVH/75/ U10562 tuf (EF-1)
Trypanosoma cruzi USAMRU-K/18Y L76077 tuf (EF-1) Human and plants
Arabidopsis thaliana Columbia X89227 tuf (EF-1) Glycine max Ceresia
X89058 tuf (EF-1) Glycine max Ceresia Y15107 tuf (EF-1) Glycine max
Ceresia Y15108 tuf (EF-1) Glycine max Maple Arrow X66062 tuf (EF-1)
Homo sapiens X03558 tuf (EF-1) Pyramimonas disomata AB008010 tuf
atpD sequences Bacteria Acetobacterium woodi DSM 1030 U10505 atpD
Actinobacillus actinomycetemcomitans HK1651 Genome project.sup.2
atpD Bacillus anthracis Ames Genome project.sup.2 atpD Bacillus
firmus OF4 M60117 atpD Bacillus megaterium QM B1551 M20255 atpD
Bacillus stearothermophilus D38058 atpD Bacillus stearothermophilus
IFO1035 D38060 atpD Bacillus subtilis 168 Z28592 atpD Bacteroides
fragilis DSM 2151 M22247 atpD Bordetella bronchiseptica RB50 Genome
project.sup.2 atpD Bordetella pertussis Tohama 1 Genome
project.sup.2 atpD Borrelia burgdorferi B31 AE001122 atpD (V)
Burkholderia cepacia DSM50181 X76877 atpD Burkholderia pseudomallei
K96243 Genome project.sup.2 atpD Campylobacter jejuni NCTC 11168
CJ11168X1 atpD Chlamydia pneumoniae Genome project.sup.2 atpD (V)
Chlamydia trachomatis MoPn Genome project.sup.2 atpD (V) Chlorobium
vibrioforme DSM 263 X76873 atpD Citrobacter freundii JEO503
AF037156 atpD Clostridium acetobutylicum ATCC 824 Genome
project.sup.2 atpD Clostridium acetobutylicum DSM 792 AF101055 atpD
Clostridium difficile 630 Genome project.sup.2 atpD Corynebacterium
diphtheriae NCTC13129 Genome project.sup.2 atpD Corynebacterium
glutamicum ASO 19 X76875 atpD Corynebacterium glutamicum MJ-233
E09634 atpD Cytophaga lytica DSM 2039 M22535 atpD Enterobacter
aerogenes DSM 30053 --.sup.3 atpD Enterococcus faecalis V583 Genome
project.sup.2 atpD (V) Enterococcus hirae M90060 atpD Enterococcus
hirae ATCC 9790 D17462 atpD (V) Escherichia coli J01594 atpD
Escherichia coli M25464 atpD Escherichia coli V00267 atpD
Escherichia coli V00311 atpD Escherichia coli K12 MG1655 L10328
atpD Flavobacterium ferrugineum DSM 13524 --.sup.3 atpD Haemophilus
actinomycetemcomitans Genome project.sup.2 atpD Haemophilus
influenzae Rd U32730 atpD Helicobacter pylori NCTC 11638 AF004014
atpD Helicobacter pylori 26695 Genome project.sup.2 atpD
Helicobacter pylori J99 Genome project.sup.2 atpD Klebsiella
pneumoniae M6H 78578 Genome project.sup.2 atpD Lactobacillus casei
DSM 20021 X64542 atpD Legionella pneumophila Philadelphia-1 Genome
project.sup.2 atpD Moorella thermoacetica ATCC 39073 U64318 atpD
Mycobacterium avium 104 Genome project.sup.2 atpD Mycobacterium
bovis AF2122/97 Genome project.sup.2 atpD Mycobacterium leprae
U15186 atpD Mycobacterium leprae Genome project.sup.2 atpD
Mycobacterium tuberculosis H37Rv Z73419 atpD Mycobacterium
tuberculosis CSU#93 Genome project.sup.2 atpD Mycoplasma
gallisepticum X64256 atpD Mycoplasma genitalium G37 U39725 atpD
Mycoplasma pneumoniae M129 U43738 atpD Neisseria gonorrhoeae FA
1090 Genome project.sup.2 atpD Neisseria meningitidis Z2491 Genome
project.sup.2 atpD Pasteurella multocida Pm70 Genome project.sup.2
atpD Pectinatus frisingensis DSM 20465 X64543 atpD Peptococcus
niger DSM 20475 X76878 atpD Pirellula marina IFAM 1313 X57204 atpD
Porphyromonas gingivalis W83 Genome project.sup.2 atpD (V)
Propionigenium modestum DSM 2376 X58461 atpD Pseudomonas aeruginosa
PAO1 Genome project.sup.2 atpD Pseudomonas putida Genome
project.sup.2 atpD Rhodobacter capsulatus B100 X99599 atpD
Rhodospirillum rubrum X02499 atpD Rickettsia prowazekii F-12
AF036246 atpD Rickettsia prowazekii Madrid Genome project.sup.2
atpD Ruminococcus albus 7ATCC AB006151 atpD Salmonella bongori
JEO4162 AF037155 atpD Salmonella bongori BR1859 AF037154 atpD
Salmonella choleraesuis S83769 AF037146 atpD subsp. arizonae
Salmonella choleraesuis u24 AF037147 atpD subsp. arizonae
Salmonella choleraesuis subsp. K228 AF037140 atpD choleraesuis
serotype Dublin Salmonella choleraesuis subsp. K771 AF037139 atpD
choleraesuis serotype Dublin Salmonella choleraesuis subsp.
Div36-86 AF037142 atpD choleraesuis serotype Infantis Salmonella
choleraesuis subsp. Div95-86 AF037143 atpD choleraesuis serotype
Tennessee Salmonella choleraesuis subsp. LT2 AF037141 atpD
choleraesuis serotype Typhimurium Salmonella choleraesuis DS210/89
AF037149 atpD subsp. diarizonae Salmonella choleraesuis JEO307
AF037148 atpD subsp. diarizonae Salmonella choleraesuis S109671
AF037150 atpD subsp. diarizonae Salmonella choleraesuis S84366
AF037151 atpD subsp. houtenae Salmonella choleraesuis S84098
AF037152 atpD subsp. houtenae Salmonella choleraesuis BR2047
AF037153 atpD subsp. indica Salmonella choleraesuis NSC72 AF037144
atpD subsp. salamae Salmonella choleraesuis S114655 AF037145 atpD
subsp. salamae Shewanella putrefaciens MR-1 Genome project.sup.2
atpD Staphylococcus aureus COL Genome project.sup.2 atpD
Stigmatella aurantiaca Sga1 X76879 atpD Streptococcus bovis JB-1
AB009314 atpD Streptococcus mutans GS-5 U31170 atpD Streptococcus
mutans UAB159 Genome project.sup.2 atpD Streptococcus pneumoniae
Type 4 Genome project.sup.2 atpD (V) Streptococcus pneumoniae Type
4 Genome project.sup.2 atpD Streptococcus pyogenes M1-GAS Genome
project.sup.2 atpD (V) Streptococcus pyogenes M1-GAS Genome
project.sup.2 atpD Streptococcus sanguinis 10904 AF001955 atpD
Streptomyces lividans 1326 Z22606 atpD Thermus thermophilus HB8
D63799 atpD (V) Thiobacillus ferrooxidans ATCC 33020 M81087 atpD
Treponema pallidum Nichols AE001228 atpD (V) Vibrio alginolyticus
X16050 atpD Vibrio cholerae N16961 Genome project.sup.2 atpD
Wolinella succinogenes DSM 1470 X76880 atpD Yersinia enterocolitica
NCTC 10460 AF037157 atpD Yersinia pestis CO-92 Genome project.sup.2
atpD Archaebacteria Archaeoglobus fulgidus DSM 4304 AE001023 atpD
(V) Halobacterium salinarum S56356 atpD (V) Haloferax volcanii WR
340 X79516 atpD Methanococcus jannaschii DSM 2661 U67477 atpD (V)
Methanosarcina barkeri DSM 800 J04836 atpD (V) Fungi Candida
albicans SC5314 Genome project.sup.2 atpD Candida tropicalis M64984
atpD (V) Kluyveromyces lactis 2359/152 U37764 atpD Neurospora
crassa X53720 atpD Saccharomyces cerevisiae M12082 atpD
Saccharomyces cerevisiae X2180-1A J05409 atpD (V)
Schizosaccharomyces pombe 972 h- S47814 atpD (V)
Schizosaccharomyces pombe 972 h- M57956 atpD Parasites Giardia
lamblia WB U18938 atpD Plasmodium falciparum 3D7 L08200 atpD (V)
Trypanosoma congolense IL3000 Z25814 atpD (V) Human and plants Homo
sapiens L09234 atpD (V) Homo sapiens M27132 atpD recA sequences
Bacteria Acetobacter aceti no. 1023 S60630 recA Acetobacter
altoacetigenes MH-24 E05290 recA Acetobacter polyoxogenes NBI 1028
D13183 recA Acholeplasma laidlawii 8195 M81465 recA Acidiphilium
facilis ATCC 35904 D16538 recA Acidothermus cellulolyticus ATCC
43068 AJ006705 recA Acinetobacter calcoaceticus BD413/ADP1 L26100
recA Actinobacillus actinomycetemcomitans HK1651 Genome
project.sup.2 recA Aeromonas salmonicida A449 U83688 recA
Agrobacterium tumefaciens C58 L07902 recA Allochromatium vinosum
AJ000677 recA Aquifex aeolicus VF5 AE000775 recA Aquifex pyrophilus
Kol5a L23135 recA Azotobacter vinelandii S96898 recA Bacillus
stearothermophilus 10 Genome project.sup.2 recA Bacillus subtilis
PB1831 U87792 recA Bacillus subtilis 168 Z99112 recA Bacteroides
fragilis M63029 recA Bifidobacterium breve NCFB 2258 AF094756 recA
Blastochloris viridis DSM 133 AF022175 recA Bordetella pertussis
165 X53457 recA Bordetella pertussis Tohama I Genome project.sup.2
recA Borrelia burgdorferi Sh-2-82 U23457 recA Borrelia burgdorferi
B31 AE001124 recA Brevibacterium flavum MJ-233 E10390 recA Brucella
abortus 2308 L00679 recA Burkholderia cepacia ATCC 17616 U70431
recA Burkholderia cepacia D90120 recA Burkholderia pseudomallei
K96243 Genome project.sup.2 recA Campylobacter fetus subsp. fetus
23D AF020677 recA Campylobacter jejuni 81-176 U03121 recA
Campylobacter jejuni NCTC 11168 AL139079 recA Chlamydia trachomatis
L2 U16739 recA Chlamydia trachomatis D/UW-3/CX AE001335 recA
Chlamydophila pneumoniae CWL029 AE001658 recA Chloroflexus
aurantiacus J-10-fl AF037259 recA Clostridium acetobutylicum M94057
recA Clostridium perfringens 13 U61497 recA Corynebacterium
diphtheriae NCTC13129 Genome project.sup.2 recA Corynebacterium
glutamicum AS019 U14965 recA Corynebacterium pseudotuberculosis
C231 U30387 recA Deinococcus radiodurans KD8301 AB005471 recA
Deinococcus radiodurans R1 U01876 recA Enterobacter agglomerans 339
L03291 recA Enterococcus faecalis OGIX M81466 recA Erwinia
carotovora X55554 recA Escherichia coli J01672 recA Escherichia
coli X55552 recA Escherichia coli K-12 AE000354 recA Frankia alni
Arl3 AJ006707 recA Gluconobacter oxydans U21001 recA Haemophilus
influenzae Rd U32687 recA Haemophilus influenzae Rd U32741 recA
Haemophilus influenzae Rd L07529 recA Helicobacter pylori 69A
Z35478 recA Helicobacter pylori 26695 AE000536 recA Helicobacter
pylori J99 AE001453 recA Klebsiella pneumoniae M6H 78578 Genome
project.sup.2 recA Lactococcus lactis ML3 M88106 recA Legionella
pneumophila X55453 recA Leptospira biflexa serovar patoc U32625
recA Leptospira interrogans serovar pomona U29169 recA
Magnetospirillum magnetotacticum MS-1 X17371 recA Methylobacillus
flagellatus MFK1 M35325 recA Methylomonas clara ATCC 31226 X59514
recA Mycobacterium avium 104 Genome project.sup.2 recA
Mycobacterium bovis AF122/97 Genome project.sup.2 recA
Mycobacterium leprae X73822 recA Mycobacterium tuberculosis H37Rv
X58485 recA Mycobacterium tuberculosis CSU#93 Genome project.sup.2
recA Mycoplasma genitalium G37 U39717 recA Mycoplasma mycoides GM9
L22073 recA Mycoplasma pneumoniae ATCC 29342 MPAE000033 recA
Mycoplasma pulmonis KD735 L22074 recA Myxococcus xanthus L40368
recA Myxococcus xanthus L40367 recA Neisseria animalis NCTC 10212
U57910 recA
Neisseria cinerea LCDC 81-176 AJ223869 recA Neisseria cinerea LNP
1646 U57906 recA Neisseria cinerea NCTC 10294 AJ223871 recA
Neisseria cinerea Vedros M601 AJ223870 recA Neisseria elongata CCUG
2131 AJ223882 recA Neisseria elongata CCUG 4165A AJ223880 recA
Neisseria elongata NCTC 10660 AJ223881 recA Neisseria elongata NCTC
11050 AJ223878 recA Neisseria elongata NHITCC 2376 AJ223877 recA
Neisseria elongata CCUG 4557 AJ223879 recA subsp. intermedia
Neisseria flava Bangor 9 AJ223873 recA Neisseria flavescens LNP 444
U57907 recA Neisseria gonorrhoeae CH95 U57902 recA Neisseria
gonorrhoeae FA19 X64842 recA Neisseria gonorrhoeae MS11 X17374 recA
Neisseria gonorrhoeae Genome project.sup.2 recA Neisseria lactamica
CCUC 7757 AJ223866 recA Neisseria lactamica CCUG 7852 Y11819 recA
Neisseria lactamica LCDC 77-143 Y11818 recA Neisseria lactamica
LCDC 80-111 AJ223864 recA Neisseria lactamica LCDC 845 AJ223865
recA Neisseria lactamica NCTC 10617 U57905 recA Neisseria lactamica
NCTC 10618 AJ223863 recA Neisseria meningitidis 44/46 X64849 recA
Neisseria meningitidis Bangor 13 AJ223868 recA Neisseria
meningitidis HF116 X64848 recA Neisseria meningitidis HF130 X64844
recA Neisseria meningitidis HF46 X64847 recA Neisseria meningitidis
M470 X64850 recA Neisseria meningitidis N94II X64846 recA Neisseria
meningitidis NCTC 8249 AJ223867 recA Neisseria meningitidis P63
X64845 recA Neisseria meningitidis S3446 U57903 recA Neisseria
meningitidis FAM18 Genome project.sup.2 recA Neisseria mucosa LNP
405 U57908 recA Neisseria mucosa Vedros M1801 AJ223875 recA
Neisseria perflava CCUG 17915 AJ223876 recA Neisseria perflava LCDC
85402 AJ223862 recA Neisseria pharyngis var. flava NCTC 4590 U57909
recA Neisseria polysaccharea CCUG 18031 Y11815 recA Neisseria
polysaccharea CCUG 24845 Y11816 recA Neisseria polysaccharea CCUG
24846 Y11814 recA Neisseria polysaccharea INS MA 3008 Y11817 recA
Neisseria polysaccharea NCTC 11858 U57904 recA Neisseria sicca NRL
30016 AJ223872 recA Neisseria subflava NRL 30017 AJ223874 recA
Paracoccus denitrificans DSM 413 U59631 recA Pasteurella multocida
X99324 recA Porphyromonas gingivalis W83 U70054 recA Prevotella
ruminicola JCM 8958 U61227 recA Proteus mirabilis pG1300 X14870
recA Proteus vulgaris X55555 recA Pseudomonas aeruginosa X05691
recA Pseudomonas aeruginosa PAM 7 X52261 recA Pseudomonas
aeruginosa PAO12 D13090 recA Pseudomonas fluorescens OE 28.3 M96558
recA Pseudomonas putida L12684 recA Pseudomonas putida PpS145
U70864 recA Rhizobium leguminosarum VF39 X59956 recA biovar viciae
Rhizobium phaseoli CNPAF512 X62479 recA Rhodobacter capsulatus J50
X82183 recA Rhodobacter sphaeroides 2.4.1 X72705 recA
Rhodopseudomonas palustris N 7 D84467 recA Rickettsia prowazekii
Madrid E AJ235273 recA Rickettsia prowazekii Madrid E U01959 recA
Serratia marcescens M22935 recA Shigella flexneri X55553 recA
Shigella sonnei KNIH104S AF101227 recA Sinorhizobium meliloti 2011
X59957 recA Staphylococcus aureus L25893 recA Streptococcus
gordonii Challis V288 L20574 recA Streptococcus mutans UA96 M81468
recA Streptococcus mutans GS-5 M61897 recA Streptococcus pneumoniae
Z17307 recA Streptococcus pneumoniae R800 Z34303 recA Streptococcus
pyogenes NZ131 U21934 recA Streptococcus pyogenes D471 M81469 recA
Streptococcus salivarius M94062 recA subsp. thermophilus
Streptomyces ambofaciens DSM 40697 Z30324 recA Streptomyces
coelicolor A3(2) AL020958 recA Streptomyces lividans TK24 X76076
recA Streptomyces rimosus R6 X94233 recA Streptomyces venezuelae
ATCC10712 U04837 recA Synechococcus sp. PR6 M29495 recA
Synechocystis sp. PCC6803 D90917 recA Thermotoga maritima L23425
recA Thermotoga maritima AE001823 recA Thermus aquaticus L20095
recA Thermus thermophilus HB8 D17392 recA Thiobacillus ferrooxidans
M26933 recA Treponema denticola Genome project.sup.2 recA Treponema
pallidum Nichols AE001243 recA Vibrio anguillarum M80525 recA
Vibrio cholerae 017 X71969 recA Vibrio cholerae 2740-80 U10162 recA
Vibrio cholerae 569B L42384 recA Vibrio cholerae M549 AF117881 recA
Vibrio cholerae M553 AF117882 recA Vibrio cholerae M645 AF117883
recA Vibrio cholerae M793 AF117878 recA Vibrio cholerae M794
AF117880 recA Vibrio cholerae M967 AF117879 recA Xanthomonas citri
XW47 AF006590 recA Xanthomonas oryzae AF013600 recA Xenorhabdus
bovienii T228/1 U87924 recA Xenorhabdus nematophilus AN6 AF127333
recA Yersinia pestis 231 X75336 recA Yersinia pestis CO-92 Genome
project.sup.2 recA Fungi, parasites, human and plants Anabaena
variabilis ATCC 29413 M29680 recA Arabidopsis thaliana U43652 recA
(Rad51) Candida albicans U39808 recA (Dmc1) Coprinus cinereus
Okayama-7 U21905 recA (Rad51) Emericella nidulans Z80341 recA
(Rad51) Gallus gallus L09655 recA (Rad51) Homo sapiens D13804 recA
(Rad51) Homo sapiens D63882 recA (Dmc1) Leishmania major Friedlin
AF062379 recA (Rad51) Leishmania major Friedlin AF062380 recA
(Dmc1) Mus musculus D58419 recA (Dmc1) Neurospora crassa 74-OR23-1A
D29638 recA (Rad51) Saccharomyces cerevisiae D10023 recA (Rad51)
Schizosaccharomyces pombe Z22691 recA (Rad51) Schizosaccharomyces
pombe 972h- AL021817 recA (Dmc1) Tetrahymena thermophila PB9R
AF064516 recA (Rad51) Trypanosoma brucei stock 427 Y13144 recA
(Rad51) Ustilago maydis U62484 recA (Rad51) Xenopus laevis D38488
recA (Rad51) Xenopus laevis D38489 recA (Rad51) *tuf indicates tuf
sequences, including tuf genes, fusA genes and fusA-tuf intergenic
spacers. tuf (C) indicates tuf sequences divergent from main
(usually A and B) copies of the elongation factor-Tu tuf (EF-1)
indicates tuf sequences of the eukaryotic type (elongation factor
1.alpha.) tuf (M) indicates tuf sequences from organellar (mostly
mitochondrial) origin atpD indicates atpD sequences of the F-type
atpD (V) indicates atpD sequences of the V-Type recA indicates recA
sequences recA (Rad51) indicates rad51 sequences or homologs recA
(Dmc1) indicates dmc1 sequences or homologs .sup.1Nucleotides
sequences published in Arch. Microbiol. 1990 153: 241-247
.sup.2These sequences are from theTIGR database
(http://www.tigr.org/tdb/tdb.html) .sup.3Nucleotides sequences
published in FEMS Microbiology Letters 1988 50: 101-106
TABLE-US-00013 TABLE 12 Bacterial species used to test the
specificity of the Staphylococcus-specific amplification primers
derived from tuf sequences. Strain Reference number Staphylococcal
species (n = 27) Staphylococcus arlettae ATCC 43957 Staphylococcus
aureus ATCC 35844 subsp. anaerobius Staphylococcus aureus ATCC
43300 subsp. aureus Staphylococcus auricularis ATCC 33753
Staphylococcus capitis ATCC 27840 subsp. capitis Staphylococcus
caprae ATCC 35538 Staphylococcus carnosus ATCC 51365 Staphylococcus
chromogenes ATCC 43764 Staphylococcus cohnii DSM 20260 subsp.
urealyticum Staphylococcus delphini ATCC 49171 Staphylococcus
epidermidis ATCC 14990 Staphylococcus equorum ATCC 43958
Staphylococcus felis ATCC 49168 Staphylococcus gallinarum ATCC
35539 Staphylococcus haemolyticus ATCC 29970 Staphylococcus hominis
ATCC 27844 Staphylococcus hyicus ATCC 11249 Staphylococcus
intermedius ATCC 29663 Staphylococcus kloosis ATCC 43959
Staphylococcus lentus ATCC 29070 Staphylococcus lugdunensis ATCC
43809 Staphylococcus saprophyticus ATCC 15305 Staphylococcus
schleiferi ATCC 49545 subsp. coagulans Staphylococcus sciuri ATCC
29060 subsp. sciuri Staphylococcus simulans ATCC 27848
Staphylococcus warneri ATCC 27836 Staphylococcus xylosus ATCC 29971
Gram-negative bacteria (n = 33) Acinetobacter baumannii ATCC 19606
Bacteroides distasonis ATCC 8503 Bacteroides fragilis ATCC 25285
Bulkholderia cepacia ATCC 25416 Bordetella pertussis ATCC 9797
Citrobacter freundii ATCC 8090 Enterobacter aerogenes ATCC 13048
Enterobacter cloacae ATCC 13047 Escherichia coli ATCC 25922
Haemophilus influenzae ATCC 8907 Haemophilus parahaemolyticus ATCC
10014 Haemophilus parainfluenzae ATCC 7901 Hafnia alvei ATCC 13337
Kingella indologenes ATCC 25869 Klebsiella oxytoca ATCC 13182
Klebsiella pneumoniae ATCC 13883 Moraxella catarrhalis ATCC 25240
Morganella morganii ATCC 25830 Neisseria gonorrhoeae ATCC 35201
Neisseria meningitidis ATCC 13077 Proteus mirabilis ATCC 25933
Proteus vulgaris ATCC 13315 Providencia rettgeri ATCC 9250
Providencia stuartii ATCC 29914 Pseudomonas aeruginosa ATCC 27853
Pseudomonas fluorencens ATCC 13525 Salmonella choleraesuis ATCC
7001 Salmonella typhimurium ATCC 14028 Serratia marcescens ATCC
8100 Shigella flexneri ATCC 12022 Shigella sonnei ATCC 29930
Stenotrophomonas maltophilia ATCC 13843 Yersinia enterocolitica
ATCC 9610 Other Gram-positive bacteria (n = 20) Bacillus subtilis
ATCC 27370 Enterococcus avium ATCC 14025 Enterococcus durans ATCC
19432 Enterococcus faecalis ATCC 19433 Enterococcus faecium ATCC
19434 Enterococcus flavescens ATCC 49996 Enterococcus gallinarum
ATCC 49573 Lactobacillus acidophilus ATCC 4356 Lactococcus lactis
ATCC 11454 Listeria innocua ATCC 33090 Listeria ivanovii ATCC 19119
Listeria monocytogenes ATCC 15313 Macrococcus caseolyticus ATCC
13548 Streptococcus agalactiae ATCC 13813 Streptococcus anginosus
ATCC 33397 Streptococcus bovis ATCC 33317 Streptococcus mutans ATCC
25175 Streptococcus pneumoniae ATCC 6303 Streptococcus pyogenes
ATCC 19615 Streptococcus salivarius ATCC 7073
TABLE-US-00014 TABLE 13 Bacterial species used to test the
specificity of the penicillin-resistant Streptococcus pneumoniae
assay. Strain Reference number Gram-positive species (n = 67)
Abiotrophia adiacens ATCC 49175 Abiotrophia defectiva ATCC 49176
Actinomyces pyogenes ATCC 19411 Bacillus anthracis ATCC 4229
Bacillus cereus ATCC 14579 Bifidobacterium breve ATCC 15700
Clostridium difficile ATCC 9689 Enterococcus avium ATCC 14025
Enterococcus casseliflavus ATCC 25788 Enterococcus dispar ATCC
51266 Enterococcus durans ATCC 19432 Enterococcus faecalis ATCC
29212 Enterococcus faecium ATCC 19434 Enterococcus flavescens ATCC
49996 Enterococcus gallinarum ATCC 49573 Enterococcus hirae ATCC
8043 Enterococcus mundtii ATCC 43186 Enterococcus raffinosus ATCC
49427 Lactobacillus lactis ATCC 19435 Lactobacillus monocytogenes
ATCC 15313 Mobiluncus curtisii ATCC 35242 Peptococcus niger ATCC
27731 Peptostreptococcus acones ATCC 6919 Peptostreptococcus
anaerobius ATCC 27337 Peptostreptococcus ATCC 2639 asaccharolyticus
Peptostreptococcus lactolyticus ATCC 51172 Peptostreptococcus
magnus ATCC 15794 Peptostreptococcus prevotii ATCC 9321
Peptostreptococcus tetradius ATCC 35098 Staphylococcus aureus ATCC
25923 Staphylococcus capitis ATCC 27840 Staphylococcus epidermidis
ATCC 14990 Staphylococcus haemolyticus ATCC 29970 Staphylococcus
hominis ATCC 27844 Staphylococcus lugdunensis ATCC 43809
Staphylococcus saprophyticus ATCC 15305 Staphylococcus simulans
ATCC 27848 Staphylococcus. warneri ATCC 27836 Streptococcus
acidominimus ATCC 51726 Streptococcus agalactiae ATCC 12403
Streptococcus anginosus ATCC 33397 Streptococcus bovis ATCC 33317
Streptococcus constellatus ATCC 27823 Streptococcus cricetus ATCC
19624 Streptococcus cristatus ATCC 51100 Streptococcus downei ATCC
33748 Streptococcus dysgalactiae ATCC 43078 Streptococcus equi ATCC
9528 Streptococcus ferus ATCC 33477 Streptococcus gordonii ATCC
10558 Streptococcus intermedius ATCC 27335 Streptococcus mitis ATCC
903 Streptococcus mitis LSPQ 2583 Streptococcus mitis ATCC 49456
Streptococcus mutans ATCC 27175 Streptococcus oralis ATCC 10557
Streptococcus oralis ATCC 9811 Streptococcus oralis ATCC 35037
Streptococcus parasanguinis ATCC 15912 Streptococcus parauberis
ATCC 6631 Streptococcus rattus ATCC 15912 Streptococcus salivarius
ATCC 7073 Streptococcus sanguinis ATCC 10556 Streptococcus suis
ATCC 43765 Streptococcus uberis ATCC 19436 Streptococcus
vestibularis ATCC 49124 Gram-negative species (n = 33)
Actinetobacter baumannii ATCC 19606 Bordetella pertussis ATCC 9797
Citrobacter diversus ATCC 27028 Citrobacter freundii ATCC 8090
Enterobacter aerogenes ATCC 13048 Enterobacter agglomerans ATCC
27155 Enterobacter cloacae ATCC 13047 Escherichia coli ATCC 25922
Haemophilus ducreyi ATCC 33940 Haemophilus haemolyticus ATCC 33390
Haemophilus influenzae ATCC 9007 Haemophilus parainfluenzae ATCC
7901 Hafnia alvei ATCC 13337 Klebsiella oxytoca ATCC 13182
Klebsiella pneumoniae ATCC 13883 Moraxella atlantae ATCC 29525
Moraxella catarrhalis ATCC 43628 Moraxella morganii ATCC 13077
Neisseria gonorrhoeae ATCC 35201 Neisseria meningitidis ATCC 13077
Proteus mirabilis ATCC 25933 Proteus vulgaris ATCC 13315
Providencia alcalifaciens ATCC 9886 Providencia rettgeri ATCC 9250
Providencia rustigianii ATCC 33673 Providencia stuartii ATCC 33672
Pseudomonas aeruginosa ATCC 35554 Pseudomonas fluorescens ATCC
13525 Pseudomonas stutzeri ATCC 17588 Salmonella typhimurium ATCC
14028 Serratia marcescens ATCC 13880 Shigella flexneri ATCC 12022
Yersina enterocolitica ATCC 9610
TABLE-US-00015 TABLE 14 Bacterial species (n = 104) detected by the
platelet contaminants assay. Abiotrophia adiacens Abiotrophia
defective Acinetobacter baumannii Acinetobacter lwoffi Aerococcus
viridans Bacillus anthracis Brucella abortus Burkholderia cepacia
Citrobacter diversus Citrobacter freundii Enterobacter aerogenes
Enterobacter agglomerans Enterococcus avium Enterococcus
casseliflavus Enterococcus dispar Enterococcus durans Enterococcus
faecalis Enterococcus faecium Enterococcus flavescens Enterococcus
gallinarum Enterococcus mundtii Enterococcus raffinosus
Enterococcus solitarius Gemella morbillorum Haemophilus ducreyi
Haemophilus haemolyticus Haemophilus influenzae Haemophilus
parahaemolyticus Haemophilus parainfluenzae Hafnia alvei Kingella
kingae Legionella pneumophila Megamonas hypermegale Moraxella
atlantae Moraxella catarrhalis Morganella morganii Neisseria
gonorrheae Neisseria meningitidis Pasteurella aerogenes Pasteurella
multocida Peptostreptococcus magnus Proteus mirabilis Providencia
alcalifaciens Providencia rettgeri Providencia rustigianii
Providencia stuartii Pseudomonas fluorescens Pseudomonas stutzeri
Salmonella bangori Salmonella enteritidis Salmonella gallinarum
Salmonella typhimurium Serratia liquefaciens Shigella flexneri
Shigella sonnei Staphylococcus capitis Staphylococcus haemolyticus
Staphylococcus hominis Staphylococcus lugdunensis Staphylococcus
saprophyticus Staphylococcus simulans Staphylococcus warneri
Stenotrophomonas maltophilia Streptococcus acidominimus
Streptococcus anginosus Streptococcus bovis Streptococcus
constellatus Streptococcus cricetus Streptococcus cristatus
Streptococcus dysgalactiae Streptococcus equi Streptococcus ferus
Streptococcus gordonii Streptococcus intermedius Streptococcus
macacae Streptococcus mitis Streptococcus oralis Streptococcus
parasanguinis Streptococcus parauberis Streptococcus pneumoniae
Streptococcus ratti Streptococcus sobrinus Streptococcus uberis
Streptococcus vestibularis Vibrio cholerae Yersinia enterocolitica
Yersinia pestis Bold characters indicate the major bacterial
contaminants found in platelet concentrates.
TABLE-US-00016 TABLE 15 Microorganisms identified by commercial
systems.sup.1. Abiotrophia adiacens (Streptococcus adjacens)
Abiotrophia defectiva (Streptococcus defectivus) Achromobacter
species Acidaminococcus fermentans Acinetobacter alcaligenes
Acinetobacter anitratus Acinetobacter baumannii Acinetobacter
calcoaceticus Acinetobacter calcoaceticus biovar anitratus
Acinetobacter calcoaceticus biovar lwoffi Acinetobacter
genomospecies Acinetobacter haemolyticus Acinetobacter johnsonii
Acinetobacter junii Acinetobacter lwoffii Acinetobacter
radioresistens Acinetobacter species Actinobacillus
actinomycetemcomitans Actinobacillus capsulatus Actinobacillus
equuli Actinobacillus hominis Actinobacillus lignieresii
Actinobacillus pleuropneumoniae Actinobacillus species
Actinobacillus suis Actinobacillus ureae Actinomyces bovis
Actinomyces israelii Actinomyces meyeri Actinomyces naeslundii
Actinomyces neuii subsp. anitratus Actinomyces neuii subsp. neuii
Actinomyces odontolyticus Actinomyces pyogenes Actinomyces radingae
Actinomyces species Actinomyces turicensis Actinomyces viscosus
Aerococcus species Aerococcus viridans Aeromonas caviae Aeromonas
hydrophila Aeromonas hydrophila group Aeromonas jandaei Aeromonas
salmonicida Aeromonas salmonicida subsp. achromogenes Aeromonas
salmonicida subsp. masoucida Aeromonas salmonicida subsp.
salmonicida Aeromonas schubertii Aeromonas sobria Aeromonas species
Aeromonas trota Aeromonas veronii Aeromonas veronii biovar sobria
Aeromonas veronii biovar veronii Agrobacterium radiobacter
Agrobacterium species Agrobacterium tumefaciens Alcaligenes
denitrificans Alcaligenes faecalis Alcaligenes odorans Alcaligenes
odorans (Alcaligenes faecalis) Alcaligenes species Alcaligenes
xylosoxidans Alcaligenes xylosoxidans subsp. denitrificans
Alcaligenes xylosoxidans subsp. xylosoxidans Alloiococcus otitis
Anaerobiospirillum succiniciproducens Anaerovibrio lipolytica
Arachnia propionica Arcanobacterium (Actinomyces) bemardiae
Arcanobacterium (Actinomyces) pyogenes Arcanobacterium haemolyticum
Arcobacter cryaerophilus (Campylobacter cryaerophila) Arthrobacter
globiformis Arthrobacter species Arxiozyma telluris (Torulopsis
pintolopesii) Atopobium minutum (Lactobacillus minutus)
Aureobacterium species Bacillus amyloliquefaciens Bacillus
anthracis Bacillus badius Bacillus cereus Bacillus circulans
Bacillus coagulans Bacillus firmus Bacillus lentus Bacillus
licheniformis Bacillus megaterium Bacillus mycoides Bacillus
pantothenticus Bacillus pumilus Bacillus species Bacillus
sphaericus Bacillus stearothermophilus Bacillus subtilis Bacillus
thuringiensis Bacteroides caccae Bacteroides capillosus Bacteroides
distasonis Bacteroides eggerthii Bacteroides fragilis Bacteroides
merdae Bacteroides ovatus Bacteroides species Bacteroides
splanchnicus Bacteroides stercoris Bacteroides thetaiotaomicron
Bacteroides uniformis Bacteroides ureolyticus (B. corrodens)
Bacteroides vulgatus Bergeyella (Weeksella) zoohelcum
Bifidobacterium adolescentis Bifidobacterium bifidum
Bifidobacterium breve Bifidobacterium dentium Bifidobacterium
infantis Bifidobacterium species Blastoschizomyces (Dipodascus)
capitatus Bordetella avium Bordetella bronchiseptica Bordetella
parapertussis Bordetella pertussis Bordetella species Borrelia
species Branhamella (Moraxella) catarrhalis Branhamella species
Brevibacillus brevis Brevibacillus laterosporus Brevibacterium
casei Brevibacterium epidermidis Brevibacterium linens
Brevibacterium species Brevundimonas (Pseudomonas) diminuta
Brevundimonas (Pseudomonas) vesicularis Brevundimonas species
Brochothrix thermosphacta Brucella abortus Brucella canis Brucella
melitensis Brucella ovis Brucella species Brucella suis Budvicia
aquatica Burkholderia (Pseudomonas) cepacia Burkholderia
(Pseudomonas) gladioli Burkholderia (Pseudomonas) mallei
Burkholderia (Pseudomonas) pseudomallei Burkholderia species
Buttiauxella agrestis Campylobacter coli Campylobacter concisus
Campylobacter fetus Campylobacter fetus subsp. fetus Campylobacter
fetus subsp. venerealis Campylobacter hyointestinalis Campylobacter
jejuni subsp. doylei Campylobacter jejuni subsp. jejuni
Campylobacter lari Campylobacter lari subsp. UPTC Campylobacter
mucosalis Campylobacter species Campylobacter sputorum
Campylobacter sputorum subsp. bubulus Campylobacter sputorum subsp.
fecalis Campylobacter sputorum subsp. sputorum Campylobacter
upsaliensis Candida (Clavispora) lusitaniae Candida (Pichia)
guilliermondii Candida (Torulopsis) glabrata Candida albicans
Candida boidinii Candida catenulata Candida ciferrii Candida
colliculosa Candida conglobata Candida curvata (Cryptococcus
curvatus) Candida dattila Candida dubliniensis Candida famata
Candida globosa Candida hellenica Candida holmii Candida humicola
Candida inconspicua Candida intermedia Candida kefyr Candida krusei
Candida lambica Candida magnoliae Candida maris Candida melibiosica
Candida membranaefaciens Candida norvegensis Candida norvegica
Candida parapsilosis Candida paratropicalis Candida pelliculosa
Candida pseudotropicalis Candida pulcherrima Candida ravautii
Candida rugosa Candida sake Candida silvicola Candida species
Candida sphaerica Candida stellatoidea Candida tenuis Candida
tropicalis Candida utilis Candida valida Candida vini Candida
viswanathii Candida zeylanoides Capnocytophaga gingivalis
Capnocytophaga ochracea Capnocytophaga species Capnocytophaga
sputigena Cardiobacterium hominis Camobacterium divergens
Camobacterium piscicola CDC group ED-2 CDC group EF4 (Pasteurella
sp.) CDC group EF-4A CDC group EF-4B CDC group EQ-Z CDC group HB-5
CDC group II K-2 CDC group IV C-2 (Bordetella-like) CDC group M5
CDC group M6 Cedecea davisae Cedecea lapagei Cedecea neteri Cedecea
species Cellulomonas (Oerskovia) turbata Cellulomonas species
Chlamydia species Chromobacterium violaceum Chryseobacterium
(Flavobacterium) indologenes Chryseobacterium (Flavobacterium)
meningosepticum Chryseobacterium gleum Chryseobacterium species
Chryseomonas indologenes Citeromyces matritensis Citrobacter
amalonaticus Citrobacter braakii Citrobacter diversus Citrobacter
farmeri Citrobacter freundii Citrobacter freundii complex
Citrobacter koseri Citrobacter sedlakii Citrobacter species
Citrobacter werkmanii Citrobacter youngae Clostridium
acetobutylicum Clostridium barati Clostridium beijerinckii
Clostridium bifermentans Clostridium botulinum Clostridium
botulinum (NP) B&F Clostridium botulinum (NP) E Clostridium
botulinum (P) A&H Clostridium botulinum (P) F Clostridium
botulinum G1 Clostridium botulinum G2 Clostridium butyricum
Clostridium cadaveris Clostridium chauvoei Clostridium
clostridiiforme Clostridium difficile Clostridium fallax
Clostridium glycolicum Clostridium hastiforme Clostridium
histolyticum Clostridium innocuum Clostridium limosum Clostridium
novyi Clostridium novyi A Clostridium paraputrificum Clostridium
perfringens Clostridium putrificum Clostridium ramosum Clostridium
septicum Clostridium sordellii Clostridium species Clostridium
sphenoides Clostridium sporogenes Clostridium subterminale
Clostridium tertium Clostridium tetani Clostridium tyrobutyricum
Comamonas (Pseudomonas) acidovorans Comamonas (Pseudomonas)
testosteroni Comamonas species Corynebacterium accolens
Corynebacterium afermentans Corynebacterium amycolatum
Corynebacterium aquaticum Corynebacterium argentoratense
Corynebacterium auris Corynebacterium bovis Corynebacterium coyleae
Corynebacterium cystitidis Corynebacterium diphtheriae
Corynebacterium diphtheriae biotype belfanti Corynebacterium
diphtheriae biotype gravis Corynebacterium diphtheriae biotype
intermedius Corynebacterium diphtheriae biotype mitis
Corynebacterium flavescens Corynebacterium glucuronolyticum
Corynebacterium glucuronolyticum- seminale Corynebacterium group A
Corynebacterium group A-4 Corynebacterium group A-5 Corynebacterium
group ANF Corynebacterium group B Corynebacterium group B-3
Corynebacterium group F Corynebacterium group F-1 Corynebacterium
group F-2 Corynebacterium group G Corynebacterium group G-1
Corynebacterium group G-2 Corynebacterium group I Corynebacterium
group I-2 Corynebacterium jeikeium (group JK) Corynebacterium
kutscheri (C. murium) Corynebacterium macginleyi Corynebacterium
minutissimum Corynebacterium pilosum Corynebacterium propinquum
Corynebacterium pseudodiphtheriticum Corynebacterium
pseudotuberculosis Corynebacterium pyogenes Corynebacterium renale
Corynebacterium renale group Corynebacterium seminale
Corynebacterium species Corynebacterium striatum (C. flavidum)
Corynebacterium ulcerans Corynebacterium urealyticum (group D2)
Corynebacterium xerosis Cryptococcus albidus Cryptococcus ater
Cryptococcus cereanus Cryptococcus gastricus Cryptococcus humicolus
Cryptococcus lactativorus Cryptococcus laurentii Cryptococcus
luteolus Cryptococcus melibiosum Cryptococcus neoformans
Cryptococcus species Cryptococcus terreus Cryptococcus
uniguttulatus Debaryomyces hansenii Debaryomyces marama
Debaryomyces polymorphus Debaryomyces species Dermabacter hominis
Dermacoccus (Micrococcus) nishinomiyaensis Dietzia species
Edwardsiella hoshinae Edwardsiella ictaluri Edwardsiella species
Edwardsiella tarda Eikenella corrodens Empedobacter brevis
(Flavobacterium breve) Enterobacter aerogenes Enterobacter
agglomerans Enterobacter amnigenus Enterobacter amnigenus asburiae
(CDC enteric group 17) Enterobacter amnigenus biogroup 1
Enterobacter amnigenus biogroup 2 Enterobacter asburiae
Enterobacter cancerogenus Enterobacter cloacae Enterobacter
gergoviae Enterobacter hormaechei Enterobacter intermedius
Enterobacter sakazakii Enterobacter species Enterobacter taylorae
Enterobacter taylorae (CDC enteric group 19) Enterococcus
(Streptococcus) cecorum Enterococcus (Streptococcus) faecalis
(Group D) Enterococcus (Streptococcus) faecium(Group D)
Enterococcus (Streptococcus) saccharolyticus Enterococcus avium
(Group D) Enterococcus casseliflavus (Steptococcus faecium subsp.
casseliflavus) Enterococcus durans (Streptococcus faecium subsp.
durans) (Group D) Enterococcus gallinarum Enterococcus hirae
Enterococcus malodoratus Enterococcus mundtii Enterococcus
raffinosus Enterococcus species Erwinia amylovora Erwinia
carotovora Erwinia carotovora subsp. atroseptica Erwinia carotovora
subsp. betavasculorum Erwinia carotovora subsp. carotovora Erwinia
chrysanthemi Erwinia cypripedii Erwinia mallotivora Erwinia
nigrifluens Erwinia quercina Erwinia rhapontici Erwinia
rubrifaciens Erwinia salicis Erwinia species Erysipelothrix
rhusiopathiae Erysipelothrix species Escherichia blattae
Escherichia coli Escherichia coli A-D Escherichia coli O157:H7
Escherichia fergusonii Escherichia hermannii Escherichia species
Escherichia vulneris Eubacterium aerofaciens Eubacterium
alactolyticum Eubacterium lentum Eubacterium limosum Eubacterium
species Ewingella americana Filobasidiella neoformans Filobasidium
floriforme Filobasidium uniguttulatum Flavimonas oryzihabitans
Flavobacterium gleum Flavobacterium indologenes Flavobacterium
odoratum Flavobacterium species Francisella novicida Francisella
philomiragia Francisella species Francisella tularensis
Fusobacterium mortiferum Fusobacterium necrogenes Fusobacterium
necrophorum Fusobacterium nucleatum Fusobacterium species
Fusobacterium varium Gaffkya species
Gardnerella vaginalis Gemella haemolysans Gemella morbillorum
Gemella species Geotrichum candidum Geotrichum fermentans
Geotrichum penicillarum Geotrichum penicillatum Geotrichum species
Gordona species Haemophilus aegyptius Haemophilus aphrophilus
Haemophilus ducreyi Haemophilus haemoglobinophilus Haemophilus
haemolyticus Haemophilus influenzae Haemophilus influenzae biotype
I Haemophilus influenzae biotype II Haemophilus influenzae biotype
III Haemophilus influenzae biotype IV Haemophilus influenzae
biotype V Haemophilus influenzae biotype VI Haemophilus influenzae
biotype VII Haemophilus influenzae biotype VIII Haemophilus
paragallinarum Haemophilus parahaemolyticus Haemophilus
parainfluenzae Haemophilus parainfluenzae biotype I Haemophilus
parainfluenzae biotype II Haemophilus parainfluenzae biotype III
Haemophilus parainfluenzae biotype IV Haemophilus parainfluenzae
biotype V Haemophilus parainfluenzae biotype VI Haemophilus
parainfluenzae biotype VII Haemophilus parainfluenzae biotype VIII
Haemophilus paraphrohaemolyticus Haemophilus paraphrophilus
Haemophilus segnis Haemophilus somnus Haemophilus species Hafnia
alvei Hanseniaspora guilliermondii Hanseniaspora uvarum
Hanseniaspora valbyensis Hansenula anomala Hansenula holstii
Hansenula polymorpha Helicobacter (Campylobacter) cinaedi
Helicobacter (Campylobacter) fennelliae Helicobacter
(Campylobacter) pylori Issatchenkia orientalis Kingella
denitrificans Kingella indologenes Kingella kingae Kingella species
Klebsiella ornithinolytica Klebsiella oxytoca Klebsiella planticola
Klebsiella pneumoniae subsp. ozaenae Klebsiella pneumoniae subsp.
pneumoniae Klebsiella pneumoniae subsp. rhinoscleromatis Klebsiella
species Klebsiella terrigena Kloeckera apiculata Kloeckera apis
Kloeckera japonica Kloeckera species Kluyvera ascorbata Kluyvera
cryocrescens Kluyvera species Kluyveromyces lactis Kluyveromyces
marxianus Kluyveromyces thermotolerans Kocuria (Micrococcus)
kristinae Kocuria (Micrococcus) rosea Kocuria(Micrococcus) varians
Koserella trabulsii Kytococcus (Micrococcus) sedentarius
Lactobacillus (Weissella) viridescens Lactobacillus A Lactobacillus
acidophilus Lactobacillus B Lactobacillus brevis Lactobacillus
buchneri Lactobacillus casei Lactobacillus casei subsp. casei
Lactobacillus casei subsp. lactosus Lactobacillus casei subsp.
rhamnosus Lactobacillus catenaformis Lactobacillus cellobiosus
Lactobacillus collinoides Lactobacillus coprophilus Lactobacillus
crispatus Lactobacillus curvatus Lactobacillus delbrueckii subsp.
bulgaricus Lactobacillus delbrueckii subsp. delbrueckii
Lactobacillus delbrueckii subsp. lactis Lactobacillus fermentum
Lactobacillus fructivorans Lactobacillus helveticus Lactobacillus
helveticus subsp. jugurti Lactobacillus jensenii Lactobacillus
lindneri Lactobacillus minutus Lactobacillus paracasei subsp.
paracasei Lactobacillus pentosus Lactobacillus plantarum
Lactobacillus salivarius Lactobacillus salivarius var. salicinius
Lactobacillus species Lactococcus diacitilactis Lactococcus
garvieae Lactococcus lactis subsp. cremoris Lactococcus lactis
subsp. diacitilactis Lactococcus lactis subsp. hordniae Lactococcus
lactis subsp. lactis Lactococcus plantarum Lactococcus
raffinolactis Leclercia adecarboxylata Legionella species
Leminorella species Leptospira species Leptotrichia buccalis
Leuconostoc (Weissella) paramesenteroides Leuconostoc carnosum
Leuconostoc citreum Leuconostoc gelidum Leuconostoc lactis
Leuconostoc mesenteroides Leuconostoc mesenteroides subsp. cremoris
Leuconostoc mesenteroides subsp. dextranicum Leuconostoc
mesenteroides subsp. mesenteroides Leuconostoc species Listeria
grayi Listeria innocua Listeria ivanovii Listeria monocytogenes
Listeria murrayi Listeria seeligeri Listeria species Listeria
welshimeri Megasphaera elsdenii Methylobacterium mesophilicum
Metschnikowia pulcherrima Microbacterium species Micrococcus luteus
Micrococcus lylae Micrococcus species Mobiluncus curtisii
Mobiluncus mulieris Mobiluncus species Moellerella wisconsensis
Moraxella (Branhamella) catarrhalis Moraxella atlantae Moraxella
bovis Moraxella lacunata Moraxella nonliquefaciens Moraxella
osloensis Moraxella phenylpyruvica Moraxella species Morganella
morganii Morganella morganii subsp. morganii Morganella morganii
subsp. sibonii Mycobacterium africanum Mycobacterium asiaticum
Mycobacterium avium Mycobacterium bovis Mycobacterium chelonae
Mycobacterium fortuitum Mycobacterium gordonae Mycobacterium
kansasii Mycobacterium malmoense Mycobacterium marinum
Mycobacterium phlei Mycobacterium scrofulaceum Mycobacterium
smegmatis Mycobacterium species Mycobacterium tuberculosis
Mycobacterium ulcerans Mycobacterium xenopi Mycoplasma fermentans
Mycoplasma hominis Mycoplasma orale Mycoplasma pneumoniae
Mycoplasma species Myroides species Neisseria cinerea Neisseria
elongata subsp. elongata Neisseria flava Neisseria flavescens
Neisseria gonorrhoeae Neisseria lactamica Neisseria meningitidis
Neisseria mucosa Neisseria perflava Neisseria polysaccharea
Neisseria saprophytes Neisseria sicca Neisseria subflava Neisseria
weaveri Neisseria weaveri (CDC group M5) Nocardia species
Ochrobactrum anthropi Oerskovia species Oerskovia xanthineolytica
Oligella (Moraxella) urethralis Oligella species Oligella
ureolytica Paenibacillus alvei Paenibacillus macerans Paenibacillus
polymyxa Pantoea agglomerans Pantoea ananas (Erwinia uredovora)
Pantoea dispersa Pantoea species Pantoea stewartii Pasteurella
(Haemophilus) avium Pasteurella aerogenes Pasteurella gallinarum
Pasteurella haemolytica Pasteurella haemolyticus Pasteurella
multocida Pasteurella multocida SF Pasteurella multocida subsp.
multocida Pasteurella multocida subsp. septica Pasteurella
pneumotropica Pasteurella species Pasteurella ureae Pediococcus
acidilactici Pediococcus damnosus Pediococcus pentosaceus
Pediococcus species Peptococcus niger
Peptococcus species Peptostreptococcus anaerobius
Peptostreptococcus asaccharolyticus Peptostreptococcus indolicus
Peptostreptococcus magnus Peptostreptococcus micros
Peptostreptococcus parvulus Peptostreptococcus prevotii
Peptostreptococcus productus Peptostreptococcus species
Peptostreptococcus tetradius Phaecoccomyces exophialiae
Photobacterium damselae Pichia (Hansenula) anomala Pichia
(Hansenula) jadinii Pichia (Hansenula) petersonii Pichia angusta
(Hansenula polymorpha) Pichia carsonii (P. vini) Pichia etchellsii
Pichia farinosa Pichia fermentans Pichia membranaefaciens Pichia
norvegensis Pichia ohmeri Pichia spartinae Pichia species
Plesiomonas shigelloides Porphyromonas asaccharolytica
Porphyromonas endodontalis Porphyromonas gingivalis Porphyromonas
levii Prevotella (Bacteroides) buccae Prevotella (Bacteroides)
buccalis Prevotella (Bacteroides) corporis Prevotella (Bacteroides)
denticola Prevotella (Bacteroides) loescheii Prevotella
(Bacteroides) oralis Prevotella (Bacteroides) disiens Prevotella
(Bacteroides) oris Prevotella bivia (Bacteroides bivius) Prevotella
intermedia (Bacteroides intermedius) Prevotella melaninogenica
(Bacteroides melaninogenicus) Prevotella ruminicola
Propionibacterium acnes Propionibacterium avidum Propionibacterium
granulosum Propionibacterium propionicum Propionibacterium species
Proteus mirabilis Proteus penneri Proteus species Proteus vulgaris
Prototheca species Prototheca wickerhamii Prototheca zopfii
Providencia alcalifaciens Providencia heimbachae Providencia
rettgeri Providencia rustigianii Providencia species Providencia
stuartii Providencia stuartii urea + Pseudomonas (Chryseomonas)
luteola Pseudomonas acidovorans Pseudomonas aeruginosa Pseudomonas
alcaligenes Pseudomonas cepacia Pseudomonas chlororaphis (P.
aureofaciens) Pseudomonas fluorescens Pseudomonas fluorescens group
Pseudomonas mendocina Pseudomonas pseudoalcaligenes Pseudomonas
putida Pseudomonas species Pseudomonas stutzeri Pseudomonas
testosteroni Pseudomonas vesicularis Pseudoramibacter (Eubacterium)
alactolyticus Psychrobacter (Moraxella) phenylpyruvicus Rahnella
aquatilis Ralstonia (Pseudomonas, Burkholderia) pickettii
Rhodococcus (Corynebacterium) equi Rhodococcus species
Rhodosporidium toruloides Rhodotorula glutinis Rhodotorula minuta
Rhodotorula mucilaginosa (R. rubra) Rhodotorula species Rickettsia
species Rothia dentocariosa Saccharomyces cerevisiae Saccharomyces
exiguus Saccharomyces kluyverii Saccharomyces species Sakaguchia
dacryoides (Rhodosporidium dacryoidum) Salmonella arizonae
Salmonella choleraesuis Salmonella enteritidis Salmonella
gallinarum Salmonella paratyphi A Salmonella paratyphi B Salmonella
pullorum Salmonella species Salmonella typhi Salmonella typhimurium
Salmonella typhisuis Salmonella/Arizona Serratia ficaria Serratia
fonticola Serratia grimesii Serratia liquefaciens Serratia
marcescens Serratia odorifera Serratia odorifera type 1 Serratia
odorifera type 2 Serratia plymuthica Serratia proteamaculans
Serratia proteamaculans subsp. proteamaculans Serratia
proteamaculans subsp. quinovora Serratia rubidaea Serratia species
Shewanella (Pseudomonas, Alteromonas) putrefaciens Shigella boydii
Shigella dysenteriae Shigella flexneri Shigella sonneri Shigella
species Sphingobacterium multivorum Sphingobacterium species
Sphingobacterium spiritivorum Sphingobacterium thalpophilum
Sphingomonas (Pseudomonas) paucimobilis Sporidiobolus salmonicolor
Sporobolomyces roseus Sporobolomyces salmonicolor Sporobolomyces
species Staphylococcus (Peptococcus) saccharolyticus Staphylococcus
arlettae Staphylococcus aureus Staphylococcus aureus (Coagulase-
negative) Staphylococcus auricularis Staphylococcus capitis
Staphylococcus capitis subsp. capitis Staphylococcus capitis subsp.
ureolyticus Staphylococcus caprae Staphylococcus carnosus
Staphylococcus caseolyticus Staphylococcus chromogenes
Staphylococcus cohnii Staphylococcus cohnii subsp. cohnii
Staphylococcus cohnii subsp. urealyticum Staphylococcus epidermidis
Staphylococcus equorum Staphylococcus gallinarum Staphylococcus
haemolyticus Staphylococcus hominis Staphylococcus hominis subsp.
hominis Staphylococcus hominis subsp. novobiosepticus
Staphylococcus hyicus Staphylococcus intermedius Staphylococcus
kloosii Staphylococcus lentus Staphylococcus lugdunensis
Staphylococcus saprophyticus Staphylococcus schleiferi
Staphylococcus sciuri Staphylococcus simulans Staphylococcus
species Staphylococcus warneri Staphylococcus xylosus
Stenotrophomonas (Xanthomonas) maltophilia Stephanoascus ciferrii
Stomatococcus mucilaginosus Streptococcus acidominimus
Streptococcus agalactiae Streptococcus agalactiae (Group B)
Streptococcus agalactiae hemolytic Streptococcus agalactiae non-
hemolytic Streptococcus alactolyticus Streptococcus anginosus
Streptococcus anginosus (Group D, nonenterococci) Streptococcus
beta-hemolytic group A Streptococcus beta-hemolytic non- group A or
B Streptococcus beta-hemolytic non- group A Streptococcus
beta-hemolytic Streptococcus bovis (Group D, nonenterococci)
Streptococcus bovis I Streptococcus bovis II Streptococcus canis
Streptococcus constellatus Streptococcus constellatus
(Streptococcus milleri I) Streptococcus constellatus (viridans
Streptococcus) Streptococcus downei Streptococcus dysgalactiae
subsp. dysgalactiae Streptococcus dysgalactiae subsp. equisimilis
Streptococcus equi (Group C/Group G Streptococcus) Streptococcus
equi subsp. equi Streptococcus equi subsp. zooepidemicus
Streptococcus equinus Streptococcus equinus (Group D,
nonenterococci) Streptococcus equisimilis Streptococcus equisimulis
(Group C/Group G Streptococcus) Streptococcus Gamma (non)-
hemolytic Streptococcus gordonii Streptococcus Group B
Streptococcus Group C Streptococcus Group D Streptococcus Group E
Streptococcus Group F Streptococcus Group G Streptococcus Group L
Streptococcus Group P Streptococcus Group U Streptococcus
intermedius Streptococcus intermedius (Streptococcus milleri II)
Streptococcus intermedius (viridans Streptococcus)
Streptococcus milleri group Streptococcus mitis Streptococcus mitis
(viridans Streptococcus) Streptococcus mitis group Streptococcus
mutans Streptococcus mutans (viridans Streptococcus) Streptococcus
oralis Streptococcus parasanguis Streptococcus pneumoniae
Streptococcus porcinus Streptococcus pyogenes Streptococcus
pyogenes (Group A) Streptococcus salivarius Streptococcus
salivarius (viridans Streptococcus) Streptococcus salivarius subsp.
salivarius Streptococcus salivarius subsp. thermophilus
Streptococcus sanguis Streptococcus sanguis I (viridans
Streptococcus) Streptococcus sanguis II Streptococcus sanguis II
(viridans Streptococcus) Streptococcus sobrinus Streptococcus
species Streptococcus suis I Streptococcus suis II Streptococcus
uberis Streptococcus uberis (viridans Streptococcus) Streptococcus
vestibularis Streptococcus zooepidemicus Streptococcus
zooepidemicus (Group C) Streptomyces somaliensis Streptomyces
species Suttonella (Kingella) indologenes Tatumella ptyseos
Tetragenococcus (Pediococcus) halophilus Torulaspora delbrueckii
(Saccharomyces rosei) Torulopsis candida Torulopsis haemulonii
Torulopsis inconspicua Treponema species Trichosporon asahii
Trichosporon asteroides Trichosporon beigelii Trichosporon cutaneum
Trichosporon inkin Trichosporon mucoides Trichosporon ovoides
Trichosporon pullulans Trichosporon species Turicella otitidis
Ureaplasma species Ureaplasma urealyticum Veillonella parvula (V.
alcalescens) Veillonella species Vibrio alginolyticus Vibrio
cholerae Vibrio damsela Vibrio fluvialis Vibrio furnissii Vibrio
harveyi Vibrio hollisae Vibrio metschnikovii Vibrio mimicus Vibrio
parahaemolyticus Vibrio species Vibrio species SF Vibrio vulnificus
Weeksella (Bergeylla) virosa Weeksella species Weeksella virosa
Williopsis (Hansenula) satumus Xanthomonas campestris Xanthomonas
species Yarrowia (Candida) lipolytica Yersinia aldovae Yersinia
enterocolitica Yersinia enterocolitica group Yersinia frederiksenii
Yersinia intermedia Yersinia intermedius Yersinia kristensenii
Yersinia pestis Yersinia pseudotuberculosis Yersinia
pseudotuberculosis SF Yersinia ruckeri Yersinia species Yokenella
regensburgei Yokenella regensburgei (Koserella trabulsii) Zygoascus
hellenicus Zygosaccharomyces species .sup.1The list includes
microorganisms that may be identified by API identification test
systems and VITEK .RTM. automated identification system from
bioMerieux Inc., or by the MicroScan .RTM.-WalkAway .RTM. automated
systems from Dade Behring. Identification relies on classical
identification methods using batteries of biochemical and other
phenotypical tests.
TABLE-US-00017 TABLE 16 tuf gene sequences obtained in our
laboratory (Example 42). GenBank Species Strain no. Gene Accession
no.* Abiotrophia adiacens ATCC49175 tuf AF124224 Enterococcus avium
ATCC14025 tufA AF124220 tufB AF274715 Enterococcus casseliflavus
ATCC25788 tufA AF274716 tufB AF274717 Enterococcus cecorum
ATCC43198 tuf AF274718 Enterococcus columbae ATCC51263 tuf AF274719
Enterococcus dispar ATCC51266 tufA AF274720 tufB AF274721
Enterococcus durans ATCC19432 tufA AF274722 tufB AF274723
Enterococcus faecalis ATCC29212 tuf AF124221 Enterococcus faecium
ATCC 19434 tufA AF124222 tufB AF274724 Enterococcus gallinarum
ATCC49573 tufA AF124223 tufB AF274725 Enterococcus hirae ATCC8043
tufA AF274726 tufB AF274727 Enterococcus malodoratus ATCC43197 tufA
AF274728 tufB AF274729 Enterococcus mundtii ATCC43186 tufA AF274730
tufB AF274731 Enterococcus pseudoavium ATCC49372 tufA AF274732 tufB
AF274733 Enterococcus raffinosus ATCC49427 tufA AF274734 tufB
AF274735 Enterococcus saccharolyticus ATCC43076 tuf AF274736
Enterococcus solitarius ATCC49428 tuf AF274737 Enterococcus
sulfureus ATCC49903 tuf AF274738 Lactococcus lactis ATCC11154 tuf
AF274745 Listeria monocytogenes ATCC15313 tuf AF274746 Listeria
seeligeri ATCC35967 tuf AF274747 Staphylococcus aureus ATCC25923
tuf AF274739 Staphylococcus epidermidis ATCC14990 tuf AF274740
Streptococcus mutans ATCC25175 tuf AF274741 Streptococcus
pneumoniae ATCC6303 tuf AF274742 Streptococcus pyogenes ATCC19615
tuf AF274743 Streptococcus suis ATCC43765 tuf AF274744
*Corresponding sequence ID NO. for the above ATCC strains are given
in table 7.
TABLE-US-00018 TABLE 17 tuf gene sequences selected from databases
for Example 42. Species Gene Accession no.* Agrobacterium
tumefaciens tufA X99673 tufB X99674 Anacystis nidulans tuf X17442
Aquifex aeolicus tufA AE000657 tufB AE000657 Bacillus
stearothermophilus tuf AJ000260 Bacillus subtilis tuf AL009126
Bacteroides fragilis tuf P33165 Borrelia burgdorferi tuf AE000783
Brevibacterium linens tuf X76863 Bulkholderia cepacia tuf P33167
Campylobacter jejuni tufB Y17167 Chlamydia pneumoniae tuf AE001363
Chlamydia trachomatis tuf M74221 Corynebacterium glutamicum tuf
X77034 Cytophaga lytica tuf X77035 Deinococcus radiodurans tuf
AE000513 Escherichia coli tufA J01690 tufB J01717 Fervidobacterium
islandicum tuf Y15788 Haemophilus influenzae tufA L42023 tufB
L42023 Helicobacter pylori tuf AE000511 Homo sapiens (Human)
EF-1.alpha. X03558 Methanococcus jannaschii EF-1.alpha. U67486
Mycobacterium leprae tuf D13869 Mycobacterium tuberculosis tuf
X63539 Mycoplasma genitalium tuf L43967 Mycoplasma pneumoniae tuf
U00089 Neisseria gonorrhoeae tufA L36380 Nicotiana tabacum
(Tobacco) EF-1.alpha. U04632 Peptococcus niger tuf X76869
Planobispora rosea tuf1 U67308 Saccharomyces cerevisiae (Yeast)
EF-1.alpha. X00779 Salmonella typhimurium tufA X55116 tufB X55117
Shewanella putrefaciens tuf P33169 Spirochaeta aurantia tuf X76874
Spirulina platensis tufA X15646 Streptomyces aureofaciens tuf1
AF007125 Streptomyces cinnamoneus tuf1 X98831 Streptomyces
coelicolor tuf1 X77039 tuf3 X77040 Streptomyces collinus tuf1
S79408 Streptomyces ramocissimus tuf1 X67057 tuf2 X67058 tuf3
X67059 Synechocystis sp. tuf AB001339 Taxeobacter ocellatus tuf
X77036 Thermotoga maritima tuf AE000512 Thermus aquaticus tuf
X66322 Thermus thermophilus tuf X06657 Thiobacillus cuprinus tuf
U78300 Treponema pallidum tuf AE000520 Wolinella succinogenes tuf
X76872 *Sequence data were obtained from GenBank, EMBL, and
SWISSPROT databases. Genes were designated as appeared in the
references.
TABLE-US-00019 TABLE 18 Nucleotide and amino acid sequence
identities of EF-Tu between different enterococci and other low G +
C gram-positive bacteria. ##STR00001## ##STR00002## The upper right
triangle represents the deduced amino acid sequence identities of
gram-positive bacterial EF-Tu; while the lower left triangle
represents the DNA sequence identities of the corresponding tuf
genes. The sequence identities between different enterococcaltufA
genes are boxed while those between enterococcaltufB genes are
shaded. ##STR00003##
TABLE-US-00020 TABLE 19 Strains analyzed in Example 43. 16S rDNA
sequence accession Taxon Strain* Strain.dagger. number Cedecea
davisae ATCC 33431.sup.T Cedecea lapagei ATCC 33432.sup.T Cedecea
neteri ATCC 33855.sup.T Citrobacter amalonaticus ATCC 25405.sup.T
CDC 9020-77.sup.T AF025370 Citrobacter braakii ATCC 43162 CDC
080-58.sup.T AF025368 Citrobacter farmeri ATCC 51112.sup.T CDC
2991-81.sup.T AF025371 Citrobacter freundii ATCC 8090.sup.T DSM
30039.sup.T AJ233408 Citrobacter koseri ATCC 27156.sup.T
Citrobacter sedlakii ATCC 51115.sup.T CDC 4696-86.sup.T AF025364
Citrobacter werkmanii ATCC 51114.sup.T CDC 0876-58.sup.T AF025373
Citrobacter youngae ATCC 29935.sup.T Edwardsiella hoshinae ATCC
33379.sup.T Edwardsiella tarda ATCC 15947.sup.T CDC 4411-68
AF015259 Enterobacter aerogenes ATCC 13048.sup.T JCM 1235.sup.T
AB004750 Enterobacter agglomerans ATCC 27989 Enterobacter amnigenus
ATCC 33072.sup.T JCM 1237.sup.T AB004749 Enterobacter asburiae ATCC
35953.sup.T JCM 6051.sup.T AB004744 Enterobacter cancerogenus ATCC
35317.sup.T Enterobacter cloacae ATCC 13047.sup.T Enterobacter
gergoviae ATCC 33028.sup.T JCM 1234.sup.T AB004748 Enterobacter
hormaechei ATCC 49162.sup.T Enterobacter sakazakii ATCC 29544.sup.T
JCM 1233.sup.T AB004746 Escherichia coli ATCC 11775.sup.T ATCC
11775.sup.T X80725 Escherichia coli ATCC 25922 ATCC 25922 X80724
Escherichia coli (ETEC) ATCC 35401 Escherichia coli (O157:H7) ATCC
43895 ATCC 43895 Z83205 Escherichia fergusonii ATCC 35469.sup.T
Escherichia hermanii ATCC 33650.sup.T Escherichia vulneris ATCC
33821.sup.T ATCC 33821.sup.T X80734 Ewingella americana ATCC
33852.sup.T NCPPB 3905 X88848 Hafnia alvei ATCC 13337.sup.T ATCC
13337.sup.T M59155 Klebsiella ornithinolytica ATCC 31898 CIP
103.364 U78182 Klebsiella oxytoca ATCC 33496 ATCC 13182.sup.T
U78183 Klebsiella planticola ATCC 33531.sup.T JCM 7251.sup.T
AB004755 Klebsiella pneumoniae subsp. pneumoniae ATCC 13883.sup.T
DSM 30104.sup.T AJ233420 subsp. ozaenae ATCC 11296.sup.T ATCC
11296.sup.T Y17654 subsp. rhinoscleromatis ATCC 13884.sup.T
Kluyvera ascorbata ATCC 33433.sup.T ATCC 14236 Y07650 Kluyvera
cryocrescens ATCC 33435.sup.T Kluyvera georgiana ATCC 51603.sup.T
Leclercia adecarboxylata ATCC 23216.sup.T Leminorella grimontii
ATCC 33999.sup.T DSM 5078.sup.T AJ233421 Moellerella wisconsensis
ATCC 35017.sup.T Morganella morganii ATCC 25830.sup.T Pantoea
agglomerans ATCC 27155.sup.T DSM 3493.sup.T AJ233423 Pantoea
dispersa ATCC 14589.sup.T Plesiomonas shigelloides ATCC 14029.sup.T
Pragia fontium ATCC 49100.sup.T DSM 5563.sup.T AJ233424 Proteus
mirabilis ATCC 25933 Proteus penneri ATCC 33519.sup.T Proteus
vulgaris ATCC 13315.sup.T DSM 30118.sup.T AJ233425 Providencia
alcalifaciens ATCC 9886.sup.T Providencia rettgeri ATCC 9250
Providencia rustigianii ATCC 33673.sup.T Providencia stuartii ATCC
33672 Rahnella aquatilis ATCC 33071.sup.T DSM 4594.sup.T AJ233426
Salmonella choleraesuis subsp. arizonae ATCC 13314.sup.T subsp.
choleraesuis serotype Choleraesuis ATCC 7001 serotype
Enteritidis.dagger-dbl. ATCC 13076.sup.T SE22 SE22 serotype
Gallinarum ATCC 9184 serotype Heidelberg ATCC 8326 serotype
Paratyphi A ATCC 9150 serotype Paratyphi B ATCC 8759 serotype
Typhi.dagger-dbl. ATCC 10749 St111 U88545 serotype
Typhimurium.dagger-dbl. ATCC 14028 serotype Virchow ATCC 51955
subsp. diarizonae ATCC 43973.sup.T subsp. houtenae ATCC 43974.sup.T
subsp. indica ATCC 43976.sup.T subsp. salamae ATCC 43972.sup.T
Serratia fonticola DSM 4576.sup.T DSM 4576.sup.T AJ233429 Serratia
grimesii ATCC 14460.sup.T DSM 30063.sup.T AJ233430 Serratia
liquefaciens ATCC 27592.sup.T Serratia marcescens ATCC 13880.sup.T
DSM 30121.sup.T AJ233431 Serratia odorifera ATCC 33077.sup.T DSM
4582.sup.T AJ233432 Serratia plymuthica DSM 4540.sup.T DSM
4540.sup.T AJ233433 Serratia rubidaea DSM 4480.sup.T DSM 4480.sup.T
AJ233436 Shigella boydii ATCC 9207 ATCC 9207 X96965 Shigella
dysenteriae ATCC 11835 ATCC 13313.sup.T X96966 ATCC 25931 X96964
Shigella flexneri ATCC 12022 ATCC 12022 X96963 Shigella sonnei ATCC
29930.sup.T Tatumella ptyseos ATCC 33301.sup.T DSM 5000.sup.T
AJ233437 Trabulsiella guamensis ATCC 49490.sup.T Yersinia
enterocolitica ATCC 9610.sup.T ATCC 9610.sup.T M59292 Yersinia
frederiksenii ATCC 33641.sup.T Yersinia intermedia ATCC 29909.sup.T
Yersinia pestis RRB KIMD27 ATCC 19428.sup.T X75274 Yersinia
pseudotuberculosis ATCC 29833.sup.T Yersinia rohdei ATCC
43380.sup.T ER-2935.sup.T X75276 Shewanella putrefaciens ATCC
8071.sup.T Vibrio cholerae ATCC 25870 ATCC 14035.sup.T X74695 T
Type strain *Strains used in this study for sequencing of partial
tuf and atpD genes. SEQ ID NOs. for tuf and atpD sequences
corresponding to the above reference strains are given in table 7.
.dagger.Strains used in other studies for sequencing of 16S rDNA
gene. When both strain numbers are on the same row, both strains
are considered to be the same although strain numbers may be
different. .dagger-dbl.Phylogenetic serotypes considered species by
the Bacteriological Code (1990 Revision).
TABLE-US-00021 TABLE 20 PCR primer pairs used in this study Primer
Nucleotide Amplicon SEQ ID NO. Sequence positions* length (bp) Tuf
664 5'-AAYATGATIACIGGIGCIGCICARATGGA- 271-299 884 3' 697
5'-CCIACIGTICKICCRCCYTCRCG-3' 1132-1156 atpD 568
5'-RTIATIGGIGCIGTIRTIGAYGT-3' 25-47 884 567
5'-TCRTCIGCIGGIACRTAIAYIGCYTG-3' 883-908 700
5'-TIRTIGAYGTCGARTTCCCTCARG-3' 38-61 871 567
5'-TCRTCIGCIGGIACRTAIAYIGCYTG-3' 883-908 *The nucleotide positions
given are for E. coli tuf and atpD sequences (GenBank accession no.
AE000410 and V00267, respectively). Numbering starts from the first
base of the initiation codon.
TABLE-US-00022 TABLE 21 Selection of M. catarrhalis-specific primer
pairs from SEQ ID NO: 29.sup.1 (466 pb DNA fragment) other than
those previously tested.sup.2. Amplicon Moraxella Moraxella size
catarrhalis catarrhalis Moraxella Primer Sequence (bp) ATCC 43628
ATCC 53879 nonliquefaciens SEQ ID NO: 118 CGCTGACGGCTTGTTTGTACCA
118 +.sup.3 + - SEQ ID NO: 119 TGTTTTGAGCTTTTTATTTTTTGA VBmcat1
TGCTTAAGATTCACTCTGCCATTTT 93 + + - VBmcat2 TAAGTCGCTGACGGCTTGTTT
VBmcat3 CCTGCACCACAAGTCATCAT 140 + + - VBmcat4
AATTCACCAACAATGTCAAAGC VBmcat5 AATGATAACCAGTCAAGCAAGC 219 + + -
VBmcat6 GGTGCATGGTGATTTGTAAAA VBmcat7 GTGTGCGTTCACTTTTACAAAT 160 +
+ - VBmcat8 GGTGTTAAGCTGATGATGAGAG VBmcat9 TGACCATGCACACCCTTATT 167
+ + - VBmcat10 TCATTGGGATGAAAGTATCGTT Moraxella Moraxella Moraxella
Moraxella Kingella Kingella Primer lacunata osloensis atlantae
phenylpyruvica indologenes kingea SEQ ID NO: 118 - - - - - - SEQ ID
NO: 119 VBmcat1 - - - - - - VBmcat2 VBmcat3 - - - - - - VBmcat4
VBmcat5 - - - - - - VBmcat6 VBmcat7 - - - - - - VBmcat8 VBmcat9 - -
- - - - VBmcat10 Neisseria Neisseria Escherichia Staphylococcus
Primer meningitidis gonorrhoeae coli aureus SEQ ID NO: 118 - - - -
SEQ ID NO: 119 VBmcat1 - - - - VBmcat2 VBmcat3 - - - - VBmcat4
VBmcat5 - - - - VBmcat6 VBmcat7 - - - - VBmcat8 VBmcat9 - - - -
VBmcat10 .sup.1SEQ ID NO. from U.S. Pat. No. 6,001,564. .sup.2All
PCR assays were performed with 1 ng of purified genomic DNA by
using an annealing temperature of 55.degree. C. and 30 cycles of
amplification. The genomic DNA from the various bacterial species
above was always isolated from reference strains obtained from
ATCC. .sup.3All position results showed a strong amplification
signal with genomic DNA from the target species M. catarrhalis.
TABLE-US-00023 TABLE 22 Selection of S. epidermidis-specific primer
pairs from SEQ ID NO: 36.sup.1 (705 pb DNA fragment) other than
those previously tested. Amplicon Staphylococcus Staphylococcus
Staphylo- Staphylo- size epidermidis, epidermidis, coccus coccus
Primer Sequence (all 25 nucleotides) (bp) ATCC 14990 ATCC 12228
capitis cohnii SEQ ID NO: 145 ATCAAAAAGTTGGCGAACCTTTTCA 125 +.sup.3
+ - - SEQ ID NO: 146 CAAAAGAGCGTGGAGAAAAGTATCA VBsep3
CATAGTCTGATTGCTCAAAGTCTTG 208 + + - - VBsep4
GCGAATAGTGAACTACATTCTGTTG + + - - VBsep5 CACGCTCTTTTGCAATTTCCATTGA
208 + + + + VBsep6 GAAGCAAATATTCAAAATGCACCAG + + + + VBsep7
AAAGTCTTTTGCTTCTTCAGATTCA 177 + + - - VBsep8
GTGTTCACAGGTATGGATGCTCTTA + + NT NT + + NT NT VBsep9
GAGCATCCATACCTGTGAACACAGA 153 + + - - VBsep10
TTTTCCAATTACAAGAGACATCAGT + + NT NT + + NT NT VBsep11
TTTGAATTCGCATGTACTTTGTTTG 135 + + - - VBsep12
CCCCGGGTTCGAAATCGATAAAAAG Staphylo- Staphylo- Staphylo- Staphylo-
Staphylo- Entero- coccus coccus Staphylo- coccus Staphylo-
Staphylo- coccus coccus Bacillus coccus Primer aureus auricularis
coccus hominis coccus coccus simulans warneri subtilis faecalis SEQ
ID NO: 145 - - - - - - - - - - SEQ ID NO: 146 VBsep3 - - + - - - -
- - - VBsep4 - - - - - - - - - - VBsep5 + - + + - - - - - - VBsep6
+ - + + - - - NT NT NT VBsep7 - - + - - - + - - - VBsep8 - NT - NT
- - + NT NT NT - NT - NT - - - NT NT NT VBsep9 - - + - + + - - - -
VBsep10 - NT + NT + - - NT NT NT - NT - NT - - - NT NT NT VBsep11 -
- - - - - - - - - VBsep12 Entero- Entero- Strepto- Strepto-
Strepto- Annealing coccus coccus Listeria coccus coccus coccus
temperature.sup.2 Primer faecium gallinarum monocytogenes
agalactiae pneumoniae pyogenes (.degree. C.) SEQ ID NO: 145 - - - -
- - 55 SEQ ID NO: 146 VBsep3 - - - - - - 55 VBsep4 - - - - - - 60
VBsep5 - - - - - - 55 VBsep6 NT NT NT NT NT NT 65 VBsep7 - - - - -
- 55 VBsep8 NT NT NT NT NT NT 60 NT NT NT NT NT NT 65 VBsep9 - - -
- - - 55 VBsep10 NT NT NT NT NT NT 60 NT NT NT NT NT NT 65 VBsep11
- - - - - - 55 VBsep12 .sup.1SEQ ID NO. from U.S. Pat. No.
6,001,564. .sup.2All PCR assays were performed with 1 ng of
purified genomic DNA by using an annealing temperature of 55 to
65.degree. C. and 30 cycles of amplification. The genomic DNA from
the various bacterial species above was always isolated from
reference strains obtained from ATCC. .sup.3All positive results
showed a strong amplification signal with genomic DNA from the
target species S. epidermidis. The instensity of the positive
amplification signal with species other than S. epidermidis was
variable. NT = NOT TESTED.
TABLE-US-00024 TABLE 23 Influence of nucleotide variation(s) on the
efficiency of the PCR amplification: Example with SEQ ID NO: 146
from S. epidermidis. Staphyloccus epidermidis.sup.2 Staphylococcus
ATCC 14990 aureus.sup.3 Sequence Number of 50.degree. C. 55.degree.
C. 50.degree. C. Primer.sup.1 (all 25 nucleotides) mutation 1 1 0.1
0.01 1 SEQ ID NO: 145 ATCAAAAAGTTGGCGAACCTTTTCA 0 SEQ ID NO: 146
CAAAAGAGCGTGGAGAAAAGTATCA 0 3+.sup.4 3+ 2+ + - VBmut1
CAAAAGAGCGTGGAGAAAAGTA CA 1 3+ 3+ 2+ + - VBmut2 CAAAAGAGCGTGGAGAAAA
TATCA 1 3+ 3+ 2+ + - VBmut3 CAAAAGAGCGTGGAGA AAGTATCA 1 3+ 3+ 2+ +
- VBmut4 CAAAAGAGCGTGG GAAAAGTATCA 1 3+ 3+ 2+ + - VBmut5 CAAAAGAGCG
GGAGAAAAGTATCA 1 3+ 3+ 2+ + - VBmut6 CAAAAGA CGTGGAGAAAAGTATCA 1 3+
3+ 2+ + - VBmut7 CAAA GAGCGTGGAGAAAAGTATCA 1 3+ 3+ 2+ + - VBmut8 C
AAAGAGCGTGGAGAAAAGTATCA 1 3+ 3+ 2+ + - VBmut9 CAAAAGAGCGTGGAGA
AAGTA CA 2 3+ 3+ 2+ + - VBmut10 CAAAAGAGCG GGAGA AAGTATCA 2 3+ 3+
2+ + - VBmut11 CAAA GAGCG GGAGAAAAGTATCA 2 3+ 3+ 2+ + - VBmut12
CAAA GAGCGTGG GAAAAGTA CA 3 3+ 3+ 2+ + - VBmut13 CAAA GAGCG GGAGA
AAGTA CA 4 3+ 2+ + - - .sup.1All PCR tests were performed with SEQ
ID NO: 145 without modification combined with SEQ ID NO: 146 or 13
modified versions of SEQ ID NO: 146. Boxed nucleotides indicate
changes in SEQ ID NO: 146. All SEQ ID NOs. are from U.S. Pat No.
6,001,564. .sup.2The tests with S. epidermidis were performed by
using an annealing temperature of 55.degree. C. with 1, 0.1 and
0.01 ng of purified genomic DNA or at 50.degree. C. with 1 ng of
purified genomic DNA. .sup.3The tests with S. aureus were performed
only at 50.degree. C. with 1 ng of genomic DNA. .sup.4The intensity
of the positive amplification signal was quantified as follows: 3+
= strong signal, 2+ = intermediate signal and + = weak signal.
TABLE-US-00025 TABLE 24 Effect of the primer length on the
efficiency of the PCR amplification.sup.1: Example with the AT-rich
SEQ ID NO: 145.sup.2 and SEQ ID NO: 146.sup.2 from S. epidermidis.
Staphylococcus Staphylo- epidermidis.sup.3 Staphylo- coccus
Staphylo- Staphylo- ATCC 14990 coccus haemo- coccus coccus Length
45.degree. C. 55.degree. C. aureus.sup.4 lyticus capitis warneri
Primer Sequence (nt) 1 0.1 0.01 1 0.1 0.01 45 55 45 55 45 55 45 55
VBsep301 ATATCATCAAAAAGTTGGCGAACCTTTTCA 30 NT NT NT 4+ 3+ 2+ NT -
NT - NT - NT - VBsep302 AATTGCAAAAGAGCGTGGAGAAAAGTATCA 30 SEQ ID
ATCAAAAAGTTGGCGAACCTTTTCA 25 .sup. 4+.sup.5 3+ 2+ 4+ 3+ 2+ - - - -
+ - - - NO: 145 SEQ ID CAAAAGAGCGTGGAGAAAAGTATCA 25 NO: 146
VBsep201 AAAGTTGGCGAACCTTTTCA 20 NT NT NT 4+ 3+ 2+ NT - NT - NT -
NT - VBsep202 GAGCGTGGAGAAAAGTATCA 20 VBsep171 GTTGGCGAACCTTTTCA 17
4+ 3+ 2+ 3+ 2+ + - - - - - - - - VBsep172 CGTGGAGAAAAGTATCA 17
VBsep151 TGGCGAACCTTTTCA 15 3+ 2+ + - - - - - - - - - - - VBsep152
TGGAGAAAAGTATCA 15 .sup.1All PCR tests were performed using an
annealing temperature of 45 or 55.degree. C. and 30 cycles of
amplification. .sup.2All SEQ ID NOs. in this Table are from U.S.
pat. No. 6,001,546. .sup.3The tests with S. epidermidis were made
with 1, 0.1 and 0.01 ng of purified genomic DNA. .sup.4The tests
with all other bacterial species were made only with 1 ng of
purified genomic DNA. .sup.5The intensity of the positive
amplification signal was quantified as follows: 4+ = very strong
signal, 3+ = strong signal, 2+ = intermediate signal and + = weak
signal. NT = not tested.
TABLE-US-00026 TABLE 25 Effect of the primer length on the
efficiency of the PCR amplification.sup.1: Example with the GC-rich
SEQ ID NO: 83.sup.2 and SEQ ID NO: 84.sup.2 from P. aeruginosa.
Pseudomonas aeruginosa.sup.3 Length ATCC 35554 Pseudomonas
Burkholderia Primer Sequence (nt) 1 0.1 0.01 fluorescens.sup.4
cepacia SEQ ID NO 83 CGAGCGGG 19 .sup. 2+.sup.5 + - - - TGGTGTTCATC
SEQ ID NO 84 CAAGTCGT 19 CGTCGGAGGGA Pse554-16a CGAGCGGG 16 2+ + -
- - TGGTGTTC Pse674-16a GTCGTCGT 16 CGGAGGGA Pse554-13b GCGGGTGG 13
2+ + - - - TGTTC Pse674-13a GTCGTCGG 13 AGGGA Shewanella
Stenotrophomonas Neisseria Haemophilus Primer putida maltophilia
meningitidis parahaemolyticus SEQ ID NO 83 - - - - SEQ ID NO 84
Pse554-16a - - - - Pse674-16a Pse554-13b - - - - Pse674-13a
.sup.1All PCR tests were performed using an annealing temperature
of 55.degree. C. and 30 cycles of amplification. .sup.2All SEQ ID
NOs. in this Table are from U.S. Pat. No. 6,001,546. .sup.3The
tests with P. aeruginosa were made with 1, 0.1 and 0.01 ng of
purified genomic DNA. .sup.4The tests with all other bacterial
species were made only with 1 ng of purified genomic DNA. .sup.5The
intensity of the positive amplification signal was quantified as
follows: 2+ = strong signal and + = moderately strong signal.
TABLE-US-00027 TABLE 26 Sequences used for the consensus of the
vanA gene GenBank Organism access number Source E. faecium M97297
E. faecium AX111560 Sequence 2293 of patent WO0123604 E. faecium
AX085668 Sequence 21 of patent WO0112803 E. faecium AX085648
Sequence 1 of patent WO0112803 E. faecium AX110408 Sequence 1141 of
Patent WO0123604 E. faecium AX110406 Sequence 1139 of patent
WO0123604 E. faecium AX110320 Sequence 1053 of patent WO0123604 E.
faecium AX110319 Sequence 1052 of patent WO0123604 E. faecium
AX110318 Sequence 1051 of patent WO0123604 E. faecium AX110316
Sequence 1049 of patent WO0123604 E. faecalis AX110321 Sequence
1054 of patent WO0123604 E. gallinarum AX110317 Sequence 1050 of
patent WO0123604 E. gallinarum AX110322 Sequence 1055 of patent
WO0123604 E. flavescens AX110324 Sequence 1057 of patent WO0123604
S. aureus AE017171.1 Direct deposit to NCBI (VRSA)
TABLE-US-00028 TABLE 27 Sequences used for the consensus of vanB
gene Organism GenBank access number Source E. faecium SLH475
AF310954.1 Tn5382 transposon ligase VanB2 (vanB2) gene E. faecium
UI709 AY145441.1 D-alanine:D-lactate ligase (vanB2) E. faecium
VRE-1 AF310953.1 Tn5382 ligase VanB2 (vanB2) gene E. faecium
U94526.1 vancomycin resistance protein B (vanB) gene, E. faecium
AJ306727.1 plasmid pVREM3123/00 partial vanHB gene and partial
vanB2 gene E. faecium AJ306726.1 plasmid pVREM2497/00 partial vanHB
gene and partial vanB2 gene E. faecium Z83305.1 vanB2, vanHB2 and
vanXB2 genes E. faecium Z83305.1 vanB2, vanHB2 and vanXB2 genes E.
faecium CG4248 AF310957.1 transposon Tn5382 ligase VanB2 (vanB2)
gene, E. faecalis L15304.1 vancomycin resistance vanB2 gene E.
faecalis U35369.1 vancomycin resistance genes, response regulator
(vanRB), protein histidine kinase (vanSB), D,D-carboxypeptidase
(vanYB), putative D-2-hydroxyacid dehydrogenase (vanHB),
D-Ala:D-Lac ligase (vanB), and putative D,D-dipeptidase (vanXB)
genes E. faecalis U00456.1 vanB gene E. faecalis L06138.1 VANB gene
E. faecalis U72704.1 vancomycin resistance protein (vanB) gene E.
faecalis T4059 AF310955.1 transposon Tn5382 ligase VanB2 (vanB2)
gene E. faecalis L15304.1 vancomycin resistance vanB2 gene E.
faecalis T4059 AF310955.1 transposon Tn5382 ligase VanB2 (vanB2)
gene E. faecalis AF192329.1 transposon Tn1549, complete sequence S.
bovis biotype II Z70527.1 partial vanB2 gene S. lutetiensis strain
5-F9 AY035703.1 Tn5382-like transposon ligase VanB2 (vanB2) gene
*Deposited directly to NCBI
TABLE-US-00029 TABLE 28 Number of positive assay results out of
five in VanR Assay. DNA copies number E. faecium E. faecalis 0.5 2
2 2.5 5 3 5 5 4 10 5 5 20 5 5
TABLE-US-00030 TABLE 29 First list of exemplary strains tested with
VanR assay. ATCC or Ref. No Strains number 1 Enterococcus
casseliflavus 25788 2 Enterococcus cecorum 43198 3 Enterococcus
columbae 51263 4 Enterococcus malodoratus 43197 5 Enterococcus
mundtii 43186 6 Enterococcus pseudoavium 49372 7 Enterococcus
raffinosus 49427 8 Enterococcus dispar 51266 9 Enterococcus durans
19432 10 Enterococcus faecalis 19433 11 Enterococcus faecalis 29212
12 Enterococcus faecalis R830 13 Enterococcus faecium 19434 14
Enterococcus faecium N97-330 15 Enterococcus flavescens 49996 16
Enterococcus gallinarum 49573 17 Enterococcus saccharolyticus 43076
18 Enterococcus sulfureus 49903 19 Enterococcus faecium (vanD)
CCRI-14889 20 Enterococcus faecium (vanD) CCRI-14889 21
Enterococcus faecalis (vanE) CCRI-1908 22 Enterococcus faecalis
(vanE) CCRI-1908 23 Enterococcus faecalis (vanG) CCRI-12848 24
Enterococcus faecalis (vanG) CCRI-12848
TABLE-US-00031 TABLE 30 Second list of exemplary strains tested
with VanR assay ATCC or Ref. No Strains number 1 Acinetobacter
twoffii CDCF 3697 2 Aeromonas hydrophila 7966 3 Bacteroides
fragilis 25285 4 Bacillus cereus 13472 5 Bifidobacterium breve
15700 6 Candida albicans 10231 7 Clostridium difficile 9689 8
Clostridium perfringens 13124 9 Corynebacterium bovis 7715 10
Escherichia coli 25922 11 Escherichia coli 23511 12 Fusobacterium
nucleatum 10953 13 Gardnerella vaginalis 14019 14 Klebsiella
oxytoca 33496 15 Klebsiella pneumoniae 13883 16 Listeria
monocytogenes L 374 17 Morganella morganii subsp. morganii 25830 18
Peptostreptococcus anaerobius 27337 19 Peptostreptococcus
asaccharolyticus LSPQ 2639 20 Porphyromonas asaccharolytica 25260
21 Prevotella melaninogenica 25845 22 Propionibacterium acnes 6919
23 Staphylococcus epidermidis 14990 24 Stenotrophomonas maltophilia
13637
TABLE-US-00032 TABLE 31 Third list of exemplary strains tested with
VanR assay. ATCC or Ref. No Strains number 1 Abiotrophia defectiva
49176 2 Acinetobacter baumannii 19606 3 Actinomyces pyogenes 19411
4 Citrobacter braakii 43162 5 Citrobacter koseri 27028 6
Corynebacterium genitalium LSPQ 3583 7 Enterobacter cloacae 13047 8
Hafnia alvei 13337 9 Homo sapiens 2.16 10 Lactobacillus acidophilus
4356 11 Lactobacillus gasseri 33323 12 Mobiluncus curtisii subsp.
holmesii 35242 13 Neisseria gonorrhoeae 35201 14 Pantoea
agglomerans 27155 15 Pseudomonas aeruginosa 35554 16 Salmonella
enterica subsp. Arizonae 13314 17 Salmonella enterica subsp.
Enterica 7001 18 Salmonella typhimurium 14028 19 Shigella flexneri
12022 20 Shigella sonnei 29930 21 Staphylococcus aureus 43300 22
Streptococcus anginosus 33397 23 Streptococcus bovis 33317 24
Vibrio cholerae 25870 25 Yersinia enterocolitica subs. enterolitica
23715
TABLE-US-00033 TABLE 32 Vancomycin resistant strains tested with
VanR assay at 10.sup.5 CFU/rx. ATCC or Ref. No Strains Resistance
gene number 1-2 Enterococcus casseliflavus vanC2 25788 (10.sup.4
CFU/rx) 3-4 Enterococcus casseliflavus vanC2 25788 5-6 Enterococcus
faecalis vanG CCRI-12849 7-8 Enterococcus faecium vanD R832 9-10
Enterococcus faecium vanD2 CCRI-15140 11-12 Enterococcus faecium
vanD CCRI-2062 13-14 Enterococcus faecium vanD CCRI-8824 15-16
Enterococcus gallinarum vanC CCRI-9133 17-18 Enterococcus
gallinarum vanC CCRI-9131 19-20 Enterococcus gallinarum vanA and
vanC1 CCRI-1561
TABLE-US-00034 TABLE 33 Composition of premix (PM) CONCENTRATION IN
FRESHLY PREPARED MIX COMPONENT (28.8 .mu.L FINAL VOLUME) FastStart
Taq DNA Polymerase 0.060 U/.mu.L MgCl.sub.2 3.010 mM Tris 100.333
mM KCl 10.033 mM (NH.sub.4).sub.2SO.sub.4 5.017 mM dNTPs 200.667
.mu.M Primer vanA649 (SEQ ID NO: 1090) 0.120 .mu.M Primer vanA754
(SEQ ID NO: 1091) 0.101 .mu.M Primer vanB626 (SEQ ID NO: 2298)
0.702 .mu.M Primer vanB774 (SEQ ID NO: 1096) 0.702 .mu.M Probe
VanA-B5c-A0 (SEQ ID NO: 2299) 0.120 .mu.M Probe Van8-850-F0 (SEW ID
NO: 2300) 0.351 .mu.M Probe Sign-B4-B0 (SEQ ID NO: 2301) 0.201
.mu.M pERVd Internal control (SEQ ID NO: 3.582 copies/.mu.L (usual)
2302) S. epidermidis DNA 895.836 copies/.mu.L BSA 0.301 mg/mL
Trehalose 4.014% *These components were added following Table
21
TABLE-US-00035 TABLE 34 Composition of final mix (MM) Sign-B4 VanA-
VanB-B50 IC (SEQ (SEQ ID NO: B5c (SEQ ID (SEQ ID NO: ID NO: n 2301)
NO: 2299) 2300) 2302) Specimen MM1-a 10 x DNA.sup.3 MM1-b 5 x
x.sup.1 TE 1X MM2-a 10 X DNA.sup.3 MM2-b 5 X x.sup.1 TE 1X MM3-a 10
x DNA.sup.3 MM3-b 5 X x.sup.1 TE 1X MM4-std 15 x X X x.sup.2
DNA.sup.4/TE 1X .sup.1At 100 times the usual concentration .sup.2At
usual concentration .sup.3At 25/50 VanA/VanB DNA copies .sup.4At 1
ng/rx VanA/VanB DNA copies
TABLE-US-00036 TABLE 35 Positive results obtained in indicated
beacon channel Sign-B4 VanB-B50 (SEQ ID NO: VanA-B5 (SEQ (SEQ ID
NO: 2301) ID NO: 2299)c 2300) TET FAM Texas Red Target MM1-a 0/5 --
-- Efm 0/5 -- -- Efs MM1-b 5/5 -- -- IC MM2-a -- 5/5 -- Efm -- 0/5
-- Efs MM2-b -- 0/5 -- IC MM3-a -- -- 0/5 Efm -- -- 5/5 Efs MM3-b
-- -- 0/5 IC MM4-std 5/5 5/5 0/5 Efm 5/5 0/5 5/5 Efs 5/5 0/5 0/5
IC
TABLE-US-00037 TABLE 36 List of enterococcal strains from different
locations around the world tested with VanR assay (dilutions
10.sup.-3). No Strains Reference number Location vanA CT vanB CT 1
Enterococcus faecalis CCRI-1471 Texas, USA 29.91 -- 2 Enterococcus
faecalis CCRI-1528 Quebec, CAN 27.05 -- 3 Enterococcus faecalis
CCRI-9741 USA -- 31.05 4 Enterococcus gallinarum CCRI-1568 Quebec,
CAN 28.60 -- 5 Enterococcus faecium CCRI-9911 Taiwan, China --
31.96 6 Enterococcus faecalis 725 Israel -- 31.55 7 Enterococcus
faecalis 1435 Brazil 28.28 -- 8 Enterococcus faecium 6169 Italy
31.63 -- 9 Enterococcus gallinarum CCRI-9737 Norway -- 29.50 10
Enterococcus faecalis CCRI-9738 Germany -- 31.35 11 Enterococcus
faecium CCRI-9740 Germany -- 29.41 12 Enterococcus faecalis 13024
Germany 27.49 -- 13 Enterococcus faecalis CCRI-9739 Germany --
31.00 14 Enterococcus faecium CCRI-9733 USA -- 31.93 15
Enterococcus faecium 1585 Argentina 29.00 -- 16 Enterococcus
faecium 715 Sweden -- 30.87 17 Enterococcus faecalis CCRI-9954
Netherlands -- 31.83 18 Enterococcus faecium CCRI-1482 Toronto, CAN
29.40 -- *All strains gave vanA or vanB positive PCR results.
TABLE-US-00038 TABLE 37 List of resistant non enterococcal strains
(vanB genotype) tested with VanR assay. Reference Texas Red CT No
Strains number (cycles) 1 Clostridium innocuum CCRI-9927 29.6 2
Clostridium sp. IDI-1987 31.9 3 Clostridium sp. CCRI-9929 32.5 4
Clostridium symbosium CCRI-9928 31.6 5 Eggerthella lenta CCRI-9926
32.4 *All strains gave vanB positive PCR results.
TABLE-US-00039 TABLE 38 VanR Assay Formulation Concentration of
Concentration of components at components at final volume (28
.mu.L) final volume (28.8 .mu.L) Raw Material Lyophilized Fresh
FastStart Taq DNA Polymerase 0..060 U/.mu.L.sup.1 0.060
U/.mu.L.sup.1 MgCl.sub.2 3.000 mM.sup.2 3.010 mM.sup.2 Tris 100.000
mM (pH 8.3).sup.2 100.333 mM (pH 8.3).sup.2 KCl 10.000 mM.sup.2
10.033 mM.sup.2 (NH.sub.4).sub.2S0.sub.4 5.000 mM.sup.2 5.017
mM.sup.2 dNTP 200.000 .mu.M 200.667 .mu.M Primer VanA 649 (SEQ ID
NO: 1090) 0.120 .mu.M 0.120 .mu.M Primer Van A 754 (SEQ ID NO:
1091) 0.100 .mu.M 0.101 .mu.M Primer Van B 626 (SEQ ID NO: 2298)
0.700 .mu.M 0.702 .mu.M Primer van B 774 (SEQ ID NO: 1096) 0.700
.mu.M 0.702 .mu.M Probe VanA-B5c-A0 (SEQ ID NO: 2299) 0.120 .mu.M
0.120 .mu.M Probe VanB-B50-F0 (SEQ ID NO: 2300) 0.350 .mu.M 0.351
.mu.M Probe Sign-B4-B0 (SEQ ID NO: 2301) 0.200 .mu.M 0.201 .mu.M
Internal control pERVd (SEQ ID NO: 2302) 3.570 copies/.mu.L 3.582
copies/.mu.L S. epidermidis DNA 892.857 copies/.mu.L.sup.3 895.836
copies/.mu.L.sup.3 BSA 0.300 mg/mL 0.301 mg/mL Trehalose 4.000%
4.014%
TABLE-US-00040 Annex I: Specific and ubiquitous primers for nucleic
acid amplification (tuf sequences). Originating DNA fragment SEQ ID
Nucleotide SEQ ID NO. Nucleotide sequence NO. position Bacterial
species: Acinetobacter baumannii 1692 5'-GGT GAG AAC TGT GGT ATC
TTA CTT 1 478-501 1693.sup.a 5'-CAT TTC AAC GCC TTC TTT CAA CTG 1
691-714 Bacterial species: Chlamydia pneumoniae 630 5'-CGG AGC TAT
CCT AGT CGT TTC A 20 2-23 629.sup.a 5'-AAG TTC CAT CTC AAC AAG GTC
AAT A 20 146-170 2085 5'-CAA ACT AAA GAA CAT ATC TTG CTA 20 45-68
2086.sup.a 5'-ATA TAA TTT GCA TCA CCT TCA AG 20 237-259 2087 5'-TCA
GCT CGT GGG ATT AGG AGA G 20 431-452 2088.sup.a 5'-AGG CTT CAC GCT
GTT AGG CTG A 20 584-605 Bacterial species: Chlamydia trachomatis
554 5'-GTT CCT TAC ATC GTT GTT TTT CTC 22 82-105 555.sup.a 5'-TCT
CGA ACT TTC TCT ATG TAT GCA 22 249-272 Parasitical species:
Cryptosporidium parvum 798 5'-TGG TTG TCC CAG CCG ATC GTT T 865
158-179 804.sup.a 5'-CCT GGG ACG GCC TCT GGC AT 865 664-683 799
5'-ACC TGT GAA TAC AAG CAA TCT 865 280-300 805.sup.a 5'-CTC TTG TCC
ATC TTA GCA GT 865 895-914 800 5'-GAT GAA ATC TTC AAC GAA GTT GAT
865 307-330 806.sup.a 5'-AGC ATC ACC AGA CTT GAT AAG 865 946-966
801 5'-ACA ACA CCG AGA AGA TCC CA 865 353-372 803.sup.a 5'-ACT TCA
GTG GTA ACA CCA GC 865 616-635 802 5'-TTG CCA TTT CTG GTT TCG TT
865 377-396 807.sup.a 5'-AAA GTG GCT TCA AAG GTT GC 865 981-1000
Bacterial species: Enterococcus faecium 1696 5'-ATG TTC CTG TAG TTG
CTG GA 64 189-208 1697.sup.a 5'-TTT CTT CAG CAA TAC CAA CAA C 64
422-443 Bacterial species: Klebsiella pneumoniae 1329 5'-TGT AGA
GCG CGG TAT CAT CAA AGT A 103 352-377 1330.sup.a 5'-AGA TTC GAA CTT
GGT GTG CGG G 103 559-571 .sup.aThese sequences are from the
complementary DNA strand of the sequence of the originating
fragment given in the Sequence Listing. Originating DNA fragment
SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO. position
Bacterial species: Mycoplasma pneumoniae 2093 5'-TGT TGG CAA TCG
AAG ACA CC 2097.sup.a 635-654 2094.sup.b 5'-TTC AAT TTC TTG ACC TAC
TTT CAA 2097.sup.a 709-732 Bacterial species: Neisseria gonorrhoeae
551 5'-GAA GAA AAA ATC TTC GAA CTG GCT A 126 256-280 552.sup.b
5'-TAC ACG GCC GGT GAC TAC G 126 378-396 2173 5'-AAG AAA AAA TCT
TCG AAC TGG CTA 126 257-280 2174.sup.b 5'-TCT ACA CGG CCG GTG 126
384-398 2175 5'-CCG CCA TAC CCC GTT T 126 654-669 2176.sup.b 5'-CGG
CAT TAC CAT TTC CAC ACC TTT 126 736-759 Bacterial species:
Pseudomonas aeruginosa 1694 5'-AAG GCA AGG ATG ACA ACG GC 153
231-250 1695.sup.b 5'-ACG ATT TCC ACT TCT TCC TGG 153 418-438
Bacterial species: Streptococcus agalactiae 549 5'-GAA CGT GAT ACT
GAC AAA CCT TTA 207-210.sup.c 308-331.sup.d 550.sup.b 5'-GAA GAA
GAA CAC CAA CGT TG 207-210.sup.c 520-539.sup.d Bacterial species:
Streptococcus pyogenes 999 5'-TTG ACC TTG TTG ATG ACG AAG AG 1002
143-165 1000.sup.b 5'-TTA GTG TGT GGG TTG ATT GAA CT 1002 622-644
1001 5'-AAG AGT TGC TTG AAT TAG TTG AG 1002 161-183 1000.sup.b
5'-TTA GTG TGT GGG TTG ATT GAA CT 1002 622-644 Parasitical species:
Trypanosoma brucei 820 5'-GAA GGA GGT GTC TGC TTA CAC 864 513-533
821.sup.b 5'-GGC GCA AAC GTC ACC ACA TCA 864 789-809 820 5'-GAA GGA
GGT GTC TGC TTA CAC 864 513-533 822.sup.b 5'-CGG CGG ATG TCC TTA
ACA GAA 864 909-929 .sup.aSequence from databases. .sup.bThese
sequences are from the complementary DNA strand of the sequence of
the originating fragment given in the Sequence Listing. .sup.cThese
sequences were aligned to derive the corresponding primer.
.sup.dThe nucleotide positions refer to the S. agalactiae tuf
sequence fragment (SEQ ID NO. 209). Originating DNA fragment SEQ ID
Nucleotide SEQ ID NO. Nucleotide sequence NO. position Parasitical
species: Trypanosoma cruzi 794 5'-GAC GAC AAG TCG GTG AAC TT
840-842.sup.a 281-300.sup.c 795.sup.b 5'-ACT TGC ACG CGA TGT GGC AG
840-842.sup.a 874-893.sup.c Bacterial genus: Clostridium sp. 796
5'-GGT CCA ATG CCW CAA ACW AGA 32,719-724, 32-52.sup.d 736.sup.a
797.sup.b 5'-CAT TAA GAA TGG YTT ATC TGT SKC TCT 32, 719-724,
320-346.sup.d 736.sup.a 808 5'-GCI TTA IWR GCA TTA GAA RAY CCA 32,
719-724, 224-247.sup.d 736.sup.a 809.sup.b 5'-TCT TCC TGT WGC AAC
TGT TCC TCT 32, 719-724, 337-360.sup.d 736.sup.a 810 5'-AGA GMW ACA
GAT AAR SCA TTC TTA 32, 719-724, 320-343.sup.d 736.sup.a 811.sup.b
5'-TRA ART AGA ATT GTG GTC TRT ATC C 32, 719-724, 686-710.sup.d
736.sup.a Bacterial genus: Corynebacterium sp. 545 5'-TAC ATC CTB
GTY GCI CTI AAC AAG TG 34-44, 662.sup.a 89-114.sup.e 546.sup.b
5'-CCR CGI CCG GTR ATG GTG AAG AT 34-44, 662.sup.a 350-372.sup.e
Bacterial genus: Enterococcus sp. 656 5'-AAT TAA TGG CTG CAG TTG
AYG A 58-72.sup.a 273-294.sup.f 657.sup.b 5'-TTG TCC ACG TTC GAT
RTC TTC A 58-72.sup.a 556-577.sup.f 656 5'-AAT TAA TGG CTG CAG TTG
AYG A 58-72.sup.a 273-294.sup.f 271.sup.b 5'-TTG TCC ACG TTG GAT
RTC TTC A 58-72.sup.a 556-577.sup.f 1137 5'-AAT TAA TGG CTG CWG TTG
AYG AA 58-72.sup.a 273-295.sup.f 1136.sup.b 5'-ACT TGT CCA CGT TSG
ATR TCT 58-72.sup.a 559-579.sup.f .sup.aThese sequences were
aligned to derive the corresponding primer. .sup.bThese sequences
are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing. .sup.c The
nucleotide positions refer to the T. cruzi tuf sequence fragment
(SEQ ID NO. 842). .sup.d The nucleotide positions refer to the C.
perfringens tuf sequence fragment (SEQ ID NO. 32). .sup.e The
nucleotide positions refer to the C. diphtheriae tuf sequence
fragment (SEQ ID NO. 662). .sup.f The nucleotide positions refer to
the E. durans tuf sequence fragment (SEQ ID NO. 61). Originating
DNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Bacterial genus: Legionella sp. 2081 5'-GRA TYR TYA AAG
TTG GTG AGG AAG 111-112.sup.a 411-434.sup.b 2082.sup.c 5'-CMA CTT
CAT CYC GCT TCG TAC C 111-112.sup.a 548-569.sup.b Bacterial genus:
Staphylococcus sp. 553 5'-GGC CGT GTT GAA CGT GGT CAA ATC A
176-203.sup.a 313-337.sup.d 575.sup.c 5'-TIA CCA TTT CAG TAC CTT
CTG GTA A 176-203.sup.a 653-677.sup.d 553 5'-GGC CGT GTT GAA CGT
GGT CAA ATC A 176-203.sup.a 313-337.sup.d 707.sup.c 5'-TWA CCA TTT
CAG TAC CTT CTG GTA A 176-203.sup.a 653-677.sup.d Bacterial genus:
Streptococcus sp. 547 5'-GTA CAG TTG CTT CAG GAC GTA TC
206-231.sup.a 372-394.sup.e 548.sup.c 5'-ACG TTC GAT TTC ATC ACG
TTG 206-231.sup.a 548-568.sup.e Fungal genus: Candida sp. 576
5'-AAC TTC RTC AAG AAG GTY GGT TAC AA 407-426, 332-357.sup.f
428-432.sup.a 632.sup.c 5'-CCC TTT GGT GGR TCS TKC TTG GA 407-426,
791-813.sup.f 428-432.sup.a 631 5'-CAG ACC AAC YGA IAA RCC ATT RAG
AT 407-426, 523-548.sup.f 428-432.sup.a 632.sup.c 5'-CCC TTT GGT
GGR TCS TKC TTG GA 407-426, 791-813.sup.f 428-432.sup.a 633 5'-CAG
ACC AAC YGA IAA RCC ITT RAG AT 407-426, 523-548.sup.f 428-432.sup.a
632.sup.c 5'-CCC TTT GGT GGR TCS TKC TTG GA 407-426, 791-813.sup.f
428-432.sup.a .sup.aThese sequences were aligned to derive the
corresponding primer. .sup.bThe nucleotide positions refer to the
L. pneumophila tuf sequence fragment (SEQ ID NO. 112). .sup.cThese
sequences are from the complementary DNA strand of the sequence of
the originating fragment given in the Sequence Listing. .sup.dThe
nucleotide positions refer to the S. aureus tuf sequence fragment
(SEQ ID NO. 179). .sup.eThe nucleotide positions refer to the S.
agalactiae tuf sequence fragment (SEQ ID NO. 209). .sup.fThe
nucleotide positions refer to the C. albicans tuf(EF-1) sequence
fragment (SEQ ID NO. 408).
Originating DNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide
sequence NO. position Fungal genus: Cryptococcus sp. 1971 5'-CYG
ACT GYG CCA TCC TYA TCA 434, 623, 1281, 150-170.sup.b 1985,
1986.sup.a 1973.sup.c 5'-RAC ACC RGI YTT GGW ITC CTT 434, 623,
1281, 464-484.sup.b 1985, 1986.sup.a 1972 5'-MGI CAG CTC ATY ITT
GCW KSC 434, 623, 1281, 260-280.sup.b 1985, 1986.sup.a 1973.sup.c
5'-RAC ACC RGI YTT GGW ITC CTT 434, 623, 1281, 464-484.sup.b 1985,
1986.sup.a Parasitical genus: Entamoeba sp. 703 5'-TAT GGA AAT TCG
AAA CAT CT 512 38-57 704.sup.c 5'-AGT GCT CCA ATT AAT GTT GG 512
442-461 703 5'-TAT GGA AAT TCG AAA CAT CT 512 38-57 705.sup.c
5'-GTA CAG TTC CAA TAC CTG AA 512 534-553 703 5'-TAT GGA AAT TCG
AAA CAT CT 512 38-57 706.sup.c 5'-TGA AAT CTT CAC ATC CAA CA 512
768-787 793 5'-TTA TTG TTG CTG CTG GTA CT 512 149-168 704.sup.c
5'-AGT GCT CCA ATT AAT GTT GG 512 442-461 Parasitical genus:
Giardia sp. 816 5'-GCT ACG ACG AGA TCA AGG GC 513 305-324 819.sup.c
5'-TCG AGC TTC TGG AGG AAG AG 513 895-914 817 5'-TGG AAG AAG GCC
GAG GAG TT 513 355-374 818.sup.c 5'-AGC CGG GCT GGA TCT TCT TC 513
825-844 Parasitical genus: Leishmania sp. 701 5'-GTG TTC ACG ATC
ATC GAT GCG 514-526.sup.a 94-114.sup.d 702.sup.c 5'-CTC TCG ATA TCC
GCG AAG CG 514-526.sup.a 913-932.sup.d .sup.aThese sequences were
aligned to derive the corresponding primer. .sup.bThe nucleotide
positions refer to the C. neoformans tuf (EF-1) sequence fragment
(SEQ ID NO. 623). .sup.cThese sequences are from the complementary
DNA strand of the sequence of the originating fragment given in the
Sequence Listing. .sup.dThe nucleotide positions refer to the L.
tropica tuf(EF-1) sequence fragment (SEQ ID NO. 526). Originating
DNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Parasitical genus: Trypanosoma sp. 823 5'-GAG CGG TAT GAY
GAG ATT GT 529, 840-842, 493-512.sup.b 864.sup.a 824.sup.c 5'-GGC
TTC TGC GGC ACC ATG CG 529, 840-842, 1171-1190.sup.b 864.sup.a
Bacterial family: Enterobacteriaceae 933 5'-CAT CAT CGT ITT CMT GAA
CAA RTG 78, 103, 146, 390-413.sup.d 168, 238, 698.sup.a 934.sup.c
5'-TCA CGY TTR RTA CCA CGC AGI AGA 78, 103, 146, 831-854.sup.d 168,
238, 698.sup.a Bacterial family: Mycobacteriaceae 539 5'-CCI TAC
ATC CTB GTY GCI CTI AAC AAG 122 85-111 540.sup.c 5'-GGD GCI TCY TCR
TCG WAI TCC TG 122 181-203 Bacterial group: Escherichia coli and
Shigella 1661 5'-TGG GAA GCG AAA ATC CTG 1668.sup.e 283-300
1665.sup.c 5'-CAG TAC AGG TAG ACT TCT G 1668.sup.e 484-502
Bacterial group: Pseudomonads group 541 5'-GTK GAA ATG TTC CGC AAG
CTG CT 153-155.sup.a 476-498.sup.f 542.sup.c 5'-CGG AAR TAG AAC TGS
GGA CGG TAG 153-155.sup.a 679-702.sup.f 541 5'-GTK GAA ATG TTC CGC
AAG CTG CT 153-155.sup.a 476-498.sup.f 544.sup.c 5'-AYG TTG TCG CCM
GGC ATT MCC AT 153-155.sup.a 749-771.sup.f .sup.aThese sequences
were aligned to derive the corresponding primer. .sup.bThe
nucleotide positions refer to the T. brucei tuf (EF-1) sequence
fragment (SEQ ID NO. 864). .sup.cThese sequences are from the
complementary DNA strand of the sequence of the originating
fragment given in the Sequence Listing. .sup.dThe nucleotide
positions refer to the E. coli tuf sequence fragment (SEQ ID NO.
698). .sup.eSequence from databases. .sup.fThe nucleotide positions
refer to the P. aeruginosa tuf sequence fragment (SEQ ID NO. 153).
Originating DNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide
sequence NO. position Parasitical group: Trypanosomatidae family
923 5'-GAC GCI GCC ATC CTG ATG ATC 511, 514-526, 166-188.sup.b 529,
840-842, 864.sup.a 924.sup.c 5'-ACC TCA GTC GTC ACG TTG GCG 511,
514-526, 648-668.sup.b 529, 840-842, 864.sup.a 925 5'-AAG CAG ATG
GTT GTG TGC TG 511, 514-526, 274-293.sup.b 529, 840-842, 864.sup.a
926.sup.c 5'-CAG CTG CTC GTG GTG CAT CTC GAT 511, 514-526,
676-699.sup.b 529, 840-842, 864.sup.a 927 5'-ACG CGG AGA AGG TGC
GCT T 511, 514-526, 389-407.sup.b 529, 840-842, 864.sup.a 928.sup.c
5'-GGT CGT TCT TCG AGT CAC CGC A 511, 514-526, 778-799.sup.b 529,
840-842, 864.sup.a Universal primers (bacteria) 636 5'-ACT GGY GTT
GAI ATG TTC CGY AA 7, 54, 78, 470-492.sup.d 100, 103, 159, 209,
224, 227.sup.b 637.sup.c 5'-ACG TCA GTI GTA CGG AAR TAG AA 7, 54,
78, 692-714.sup.d 100, 103, 159, 209, 224, 227.sup.b 638 5'-CCA ATG
CCA CAA ACI CGT GAR CAC AT 7, 54, 78, 35-60.sup.e 100, 103, 159,
209, 224, 227.sup.b 639.sup.c 5'-TTT ACG GAA CAT TTC WAC ACC WGT
IAC A 7, 54, 78, 469-496.sup.e 100, 103, 159, 209, 224, 227.sup.b
.sup.aThese sequences were aligned to derive the corresponding
primer. .sup.bThe nucleotide positions refer to the L. tropica tuf
(EF-1) sequence fragment (SEQ ID NO. 526). .sup.cThese sequences
are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing. .sup.dThe
nucleotide positions refer to the E. coli tuf sequence fragment
(SEQ ID NO. 78). .sup.eThe nucleotide positions refer to the B.
cereus tuf sequence fragment (SEQ ID NO. 7). Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Universal primer (bacteria) (continued) 643 5'-ACT GGI GTI
GAR ATG TTC CGY AA 1, 3, 4, 7, 12, 470-492.sup.b 13, 16, 49, 54,
72, 78, 85, 88, 91, 94, 98, 103, 108, 112, 115, 116, 120, 121, 126,
128, 134, 136, 146, 154, 159, 179, 186, 205, 209, 212, 224,
238.sup.a 644.sup.c 5'-ACG TCI GTI GTI CKG AAR TAG AA same as SEQ
692-714.sup.b ID NO. 643 643 5'-ACT GGI GTI GAR ATG TTC CGY AA 1,
3, 4, 7, 12, 470-492.sup.b 13, 16, 49, 54, 72, 78, 85, 88, 91, 94,
98, 103, 108, 112, 115, 116, 120, 121, 126, 128, 134, 136, 146,
154, 159, 179, 186, 205, 209, 212, 224, 238.sup.a 645.sup.c 5'-ACG
TCI GTI GTI CKG AAR TAR AA same as SEQ 692-714.sup.b ID NO. 643 646
5'-ATC GAC AAG CCI TTC YTI ATG SC 2, 13, 82 317-339.sup.d 122,
145.sup.a 647.sup.c 5'-ACG TCC GTS GTR CGG AAG TAG AAC TG 2, 13, 82
686-711.sup.d 122, 145.sup.a 646 5'-ATC GAC AAG CCI TTC YTI ATG SC
2, 13, 82 317-339.sup.d 122, 145.sup.a 648.sup.c 5'-ACG TCS GTS GTR
CGG AAG TAG AAC TG 2, 13, 82 686-711.sup.d 122, 145.sup.a
.sup.aThese sequences were aligned to derive the corresponding
primer. .sup.bThe nucleotide positions refer to the E. coli tuf
sequence fragment (SEQ ID NO. 78). .sup.cThese sequences are from
the complementary DNA strand of the sequence of the originating
fragment given in the Sequence Listing. .sup.dThe nucleotide
positions refer to the A. meyeri tuf sequence fragment (SEQ ID NO.
2) Originating DNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide
sequence NO. position Universal primer (bacteria) (continued) 649
5'-GTC CTA TGC CTC ARA CWC GIG AGC AC 8, 86, 141, 143.sup.a
33-58.sup.b 650.sup.c 5'-TTA CGG AAC ATY TCA ACA CCI GT 8, 86, 141,
143.sup.a 473-495.sup.b 636 5'-ACT GGY GTT GAI ATG TTC CGY AA 8,
86, 141, 143.sup.a 473-495.sup.b 651.sup.c 5'-TGA CGA CCA CCI TCY
TCY TTY TTC A 8, 86, 141, 143.sup.a 639-663.sup.b Universal primers
(fungi) 1974 5'-ACA AGG GIT GGR MSA AGG AGA C 404, 405, 433,
443-464.sup.d 445, 898, 1268, 1276, 1986.sup.a
1975.sup.c 5'-TGR CCR GGG TGG TTR AGG ACG 404, 405, 433,
846-866.sup.d 445, 898, 1268, 1276, 1986.sup.a 1976 5'-GAT GGA YTC
YGT YAA ITG GGA 407-412, 286-306.sup.e 414-426, 428-431, 439, 443,
447, 448, 622, 624, 665, 1685, 1987-1990.sup.a 1978.sup.c 5'-CAT
CIT GYA ATG GYA ATC TYA AT same as SEQ 553-575.sup.e ID NO. 1976
1977 5'-GAT GGA YTC YGT YAA RTG GGA same as SEQ 286-306.sup.e ID
NO. 1976 1979.sup.c 5'-CAT CYT GYA ATG GYA ASC TYA AT same as SEQ
553-575.sup.e ID NO. 1976 1981 5'-TGG ACA CCI SCA AGI GGK CYG
401-405, 281-301.sup.d 433, 435, 436, 438, 444, 445, 449, 453, 455,
457, 779, 781-783, 785, 786, 788-790, 897-903, 1267-1272,
1274-1280, 1282-1287, 1991-1998.sup.a 1980.sup.c 5'-TCR ATG GCI TCI
AIR AGR GTY T same as SEQ 488-509.sup.d ID NO. 1981 .sup.aThese
sequences were aligned to derive the corresponding primer.
.sup.bThe nucleotide positions refer to the B. distasonis tuf
sequence fragment (SEQ ID NO. 8). .sup.cThese sequences are from
the complementary DNA strand of the sequence of the originating
fragment given in the Sequence Listing. .sup.dThe nucleotide
positions refer to the A. fumigatus tuf (EF-1) sequence fragment
(SEQ ID NO. 404). .sup.eThe nucleotide positions refer to the C.
albicans tuf (EF-1) sequence fragment (SEQ ID NO. 407). Originating
DNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Universal primers (fungi) (continued) 1982 5'-TGG ACA CYI
SCA AGI GGK CYG same as SEQ 281-301.sup.a ID NO. 1981 1980.sup.b
5'-TCR ATG GCI TCI AIR AGR GTY T same as SEQ 488-509.sup.a ID NO.
1981 1983 5'-CYG AYT GCG CYA TIC TCA TCA same as SEQ 143-163.sup.a
ID NO. 1981 1980.sup.b 5'-TCR ATG GCI TCI AIR AGR GTY T same as SEQ
488-509.sup.a ID NO. 1981 1984 5'-CYG AYT GYG CYA TYC TSA TCA same
as SEQ 143-163.sup.a ID NO. 1981 1980.sup.b 5'-TCR ATG GCI TCI AIR
AGR GTY T same as SEQ 488-509.sup.a ID NO. 1981 Sequencing primers
556 5'-CGG CGC NAT CYT SGT TGT TGC 668.sup.c 306-326 557.sup.b
5'-CCM AGG CAT RAC CAT CTC GGT G 668.sup.c 1047-1068 694 5'-CGG CGC
IAT CYT SGT TGT TGC 668.sup.c 306-326 557.sup.b 5'-CCM AGG CAT RAC
CAT CTC GGT G 668.sup.c 1047-1068 664 5'-AAY ATG ATI ACI GGI GCI
GCI CAR ATG GA 619.sup.c 604-632 652.sup.b 5'-CCW AYA GTI YKI CCI
CCY TCY CTI ATA 619.sup.c 1482-1508 664 5'-AAY ATG ATI ACI GGI GCI
GCI CAR ATG GA 619.sup.c 604-632 561.sup.b 5'-ACI GTI CGG CCR CCC
TCA CGG AT 619.sup.c 1483-1505 543 5'-ATC TTA GTA GTT TCT GCT GCT
GA 607 8-30 660.sup.b 5'-GTA GAA TTG AGG ACG GTA GTT AG 607 678-700
658 5'-GAT YTA GTC GAT GAT GAA GAA TT 621 116-138 659.sup.b 5'-GCT
TTT TGI GTT TCW GGT TTR AT 621 443-465 658 5'-GAT YTA GTC GAT GAT
GAA GAA TT 621 116-138 661.sup.b 5'-GTA GAA YTG TGG WCG ATA RTT RT
621 678-700 558 5'-TCI TTY AAR TAY GCI TGG GT 665.sup.c 157-176
559.sup.b 5'-CCG ACR GCR AYI GTY TGI CKC AT 665.sup.c 1279-1301 813
5'-AAT CYG TYG AAA TGC AYC ACG A 665.sup.c 687-708 559.sup.b 5'-CCG
ACR GCR AYI GTY TGI CKC AT 665.sup.c 1279-1301 .sup.aThe nucleotide
positions refer to the A. fumigatus tuf (EF-1) sequence fragment
(SEQ ID NO. 404). .sup.bThese sequences are from the complementary
DNA strand of the sequence of the originating fragment given in the
Sequence Listing. .sup.cSequences from databases. Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Sequencing primers (continued) 558 5'-TCI TTY AAR TAY GCI
TGG GT 665.sup.a 157-176 815.sup.b 5'-TGG TGC ATY TCK ACR GAC TT
665.sup.a 686-705 560 5'-GAY TTC ATY AAR AAY ATG ATY AC 665.sup.a
289-311 559.sup.b 5'-CCG ACR GCR AYI GTY TGI CKC AT 665.sup.a
1279-1301 653 5'-GAY TTC ATI AAR AAY ATG AT 665.sup.a 289-308
559.sup.b 5'-CCG ACR GCR AYI GTY TGI CKC AT 665.sup.a 1279-1301 558
5'-TCI TTY AAR TAY GCI TGG GT 665.sup.a 157-176 655.sup.b 5'-CCR
ATA CCI CMR ATY TTG TA 665.sup.a 754-773 654 5'-TAC AAR ATY KGI GGT
ATY GG 665.sup.a 754-773 559.sup.b 5'-CCG ACR GCR AYI GTY TGI CKC
AT 665.sup.a 1279-1301 696 5'-ATI GGI CAY RTI GAY CAY GGI AAR AC
698.sup.a 52-77 697.sup.b 5'-CCI ACI GTI CKI CCR CCY TCR CG
698.sup.a 1132-1154 911 5'-GAC GGM KKC ATG CCG CAR AC 853 22-41
914.sup.b 5'-GAA RAG CTG CGG RCG RTA GTG 853 700-720 912 5'-GAC GGC
GKC ATG CCG CAR AC 846 20-39 914.sup.b 5'-GAA RAG CTG CGG RCG RTA
GTG 846 692-712 913 5'-GAC GGY SYC ATG CCK CAG AC 843 251-270
915.sup.b 5'-AAA CGC CTG AGG RCG GTA GTT 843 905-925 916 5'-GCC GAG
CTG GCC GGC TTC AG 846 422-441 561.sup.b 5'-ACI GTI CGG CCR CCC TCA
CGG AT 619.sup.a 1483-1505 664 5'-AAY ATG ATI ACI GGI GCI GCI CAR
ATG GA 619.sup.a 604-632 917.sup.b 5'-TCG TGC TAC CCG TYG CCG CCA T
846 593-614 .sup.aSequences from databases. .sup.bThese sequences
are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing. Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Sequencing primers (continued) 1221 5'-GAY ACI CCI GGI CAY
GTI GAY TT 1230.sup.a 292-314 1226.sup.b 5'-GTI RMR TAI CCR AAC ATY
TC 1230.sup.a 2014-2033 1222 5'-ATY GAY ACI CCI GGI CAY GTI GAY TT
1230.sup.a 289-314 1223.sup.b 5'-AYI TCI ARR TGI ARY TCR CCC ATI CC
1230.sup.a 1408-1433 1224 5'-CCI GYI HTI YTI GAR CCI ATI ATG
1230.sup.a 1858-1881 1225.sup.b 5'-TAI CCR AAC ATY TCI SMI ARI GGI
AC 1230.sup.a 2002-2027 1227 5'-GTI CCI YTI KCI GAR ATG TTY GGI TA
1230.sup.a 2002-2027 1229.sup.b 5'-TCC ATY TGI GCI GCI CCI GTI ATC
AT 698.sup.a 4-29 1228 5'-GTI CCI YTI KCI GAR ATG TTY GGI TAY GC
1230.sup.a 2002-2030 1229.sup.b 5'-TCC ATY TGI GCI GCI CCI GTI ATC
AT 698.sup.a 4-29 1999 5'-CAT GTC AAY ATT GGT ACT ATT GGT CAT GT
498-500, 25-53.sup.d 502, 505, 506, 508, 619, 2004, 2005.sup.c
2000.sup.b 5'-CCA CCY TCI CTC AMG TTG AAR CGT T same as SEQ
1133-1157.sup.d ID NO. 1999 2001 5'-ACY ACI TTR ACI GCY GCY ATY AC
same as SEQ 67-89.sup.d ID NO. 1999 2003.sup.b 5'-CAT YTC RAI RTT
GTC ACC TGG same as SEQ 1072-1092.sup.d ID NO. 1999 2002 5'-CCI GAR
GAR AGA GCI MGW GGT same as SEQ 151-171.sup.d ID NO. 1999
2003.sup.b 5'-CAT YTC RAI RTT GTC ACC TGG same as SEQ
1072-1092.sup.d ID NO. 1999 .sup.aSequences from databases.
.sup.bThese sequences are from the complementary DNA strand of the
sequence of the originating fragment given in the Sequence Listing.
.sup.cThese sequences were aligned to derive the corresponding
primer. .sup.dThe nucleotide positions refer to the C. albicans tuf
sequence fragment (SEQ ID NO. 2004).
TABLE-US-00041 Annex II: Specific and ubiquitous primers for
nucleic acid amplification (atpD sequences). Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Bacterial species: Acinetobacter baumannii 1690 5'-CAG GTC
CTG TTG CGA CTG AAG AA 243 186-208 1691.sup.b 5'-CAC AGA TAA ACC
TGA GTG TGC TTT C 243 394-418 Bacterial species: Bacteroides
fragilis 2134 5'-CGC GTG AAG CTT CTG TG 929 184-200 2135.sup.b
5'-TCT CGC CGT TAT TCA GTT TC 929 395-414 Bacterial species:
Bordetella pertussis 2180 5'-TTC GCC GGC GTG GGC 1672.sup.c 544-558
2181.sup.b 5'-AGC GCC ACG CGC AGG 1672.sup.c 666-680 Bacterial
species: Enterococcus faecium 1698 5'-GGA ATC AAC AGA TGG TTT ACA
AA 292 131-153 1699.sup.b 5'-GCA TCT TCT GGG AAA GGT GT 292 258-277
1700 5'-AAG ATG CGG AAA GAA GCG AA 292 271-290 1701.sup.b 5'-ATT
ATG GAT CAG TTC TTG GAT CA 292 439-461 Bacterial species:
Klebsiella pneumoniae 1331 5'-GCC CTT GAG GTA CAG AAT GGT AAT GAA
GTT 317 88-118 1332.sup.b 5'-GAC CGC GGC GCA GAC CAT CA 317 183-203
.sup.aThese sequences were aligned to derive the corresponding
primer. .sup.bThese sequences are from the complementary DNA strand
of the sequence of the originating fragment given in the Sequence
Listing. .sup.cSequence from databases. Originating DNA fragment
SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO. position
Bacterial species: Streptococcus agalactiae 627 5'-ATT GTC TAT AAA
AAT GGC GAT AAG TC 379-383.sup.a 42-67.sup.b 625.sup.c 5'-CGT TGA
AGA CAC GAC CCA AAG TAT CC 379-383.sup.a 206-231.sup.b 628 5'-AAA
ATG GCG ATA AGT CAC AAA AAG TA 379-383.sup.a 52-77.sup.b 625.sup.c
5'-CGT TGA AGA CAC GAC CCA AAG TAT CC 379-383.sup.a 206-231.sup.b
627 5'-ATT GTC TAT AAA AAT GGC GAT AAG TC 379-383.sup.a 42-67.sup.b
626.sup.c 5'-TAC CAC CTT TTA AGT AAG GTG CTA AT 379-383.sup.a
371-396.sup.b 628 5'-AAA ATG GCG ATA AGT CAC AAA AAG TA
379-383.sup.a 52-77.sup.b 626.sup.c 5'-TAC CAC CTT TTA AGT AAG GTG
CTA AT 379-383.sup.a 371-396.sup.b Bacterial group: Campylobacter
jejuni and C. coli 2131 5'-AAG CMA TTG TTG TAA ATT TTG AAA G 1576,
1600, 7-31.sup.e 1849, 1863, 2139.sup.d,a 2132.sup.c 5'-TCA TAT CCA
TAG CAA TAG TTC TA 1576, 1600, 92-114.sup.e 1849, 1863,
2139.sup.d,a Bacterial genus: Bordetella sp. 825 5'-ATG AGC ARC GSA
ACC ATC GTT CAG TG 1672.sup.d 1-26 826.sup.c 5'-TCG ATC GTG CCG ACC
ATG TAG AAC GC 1672.sup.d 1342-1367 Fungal genus: Candida sp. 634
5'-AAC ACY GTC AGR RCI ATT GCY ATG GA 460-472, 101-126.sup.f
474-478.sup.a 635.sup.c 5'-AAA CCR GTI ARR GCR ACT CTI GCT CT
460-472, 617-642.sup.f 474-478.sup.a .sup.aThese sequences were
aligned to derive the corresponding primer. .sup.bThe nucleotide
positions refer to the S. agalactiae atpD sequence fragment (SEQ ID
NO. 380). .sup.cThese sequences are from the complementary DNA
strand of the sequence of the originating fragment given in the
Sequence Listing. .sup.dSequence from databases. .sup.eThe
nucleotide positions refer to the C. jejuni atpD sequence fragment
(SEQ ID NO. 1576). .sup.fThe nucleotide positions refer to the C.
albicans atpD sequence fragment (SEQ ID NO. 460). Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Universal primers 562 5'-CAR ATG RAY GAR CCI CCI GGI GYI
MGI ATG 243, 244, 262, 528-557.sup.b 264, 280, 284, 291, 297, 309,
311, 315, 317, 324, 329, 332, 334-336, 339, 342, 343, 351, 356,
357, 364-366, 370, 375, 379, 393.sup.a 563.sup.c 5'-GGY TGR TAI CCI
ACI GCI GAI GGC AT 243, 244, 262, 687-712.sup.b 264, 280, 284, 291,
297, 309, 311, 315, 317, 324, 329, 332, 334-336, 339, 342, 343,
351, 356, 357, 364-366, 370, 375, 379, 393.sup.a 564 5'-TAY GGI CAR
ATG AAY GAR CCI CCI GGI AA 243, 244, 262, 522-550.sup.b 264, 280,
284, 291, 297, 309, 311, 315, 317, 324, 329, 332, 334-336, 339,
342, 343, 351, 356, 357, 364-366, 370, 375, 379, 393.sup.a
565.sup.c 5'-GGY TGR TAI CCI ACI GCI GAT GGD AT 243, 244, 262,
687-712.sup.b 264, 280, 284, 291, 297, 309, 311, 315, 317, 324,
329, 332, 334-336, 339, 342, 343, 351, 356, 357, 364-366, 370, 375,
379, 393.sup.a .sup.aThese sequences were aligned to derive the
corresponding primer. .sup.bThe nucleotide positions refer to the
K. pneumoniae atpD sequence fragment (SEQ ID NO. 317). .sup.cThese
sequences are from the complementary DNA strand of the sequence of
the originating fragment given in the Sequence Listing. Originating
DNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Universal primers (continued) 640 5'-TCC ATG GTI TWY GGI
CAR ATG AA 248, 284, 315, 513-535.sup.b 317, 343, 357, 366, 370,
379, 393.sup.a 641.sup.c 5'-TGA TAA CCW ACI GCI GAI GGC ATA CG 248,
284, 315, 684-709.sup.b 317, 343, 357, 366, 370, 379, 393.sup.a 642
5'-GGC GTI GGI GAR CGI ACI CGT GA 248, 284, 315, 438-460.sup.b 317,
343, 357, 366, 370, 379, 393.sup.a 641.sup.c 5'-TGA TAA CCW ACI GCI
GAI GGC ATA CG 248, 284, 315, 684-709.sup.b 317, 343, 357, 366,
370, 379, 393.sup.a Sequencing primers 566 5'-TTY GGI GGI GCI GGI
GTI GGI AAR AC 669.sup.d 445-470 567.sup.c 5'-TCR TCI GCI GGI ACR
TAI AYI GCY TG 669.sup.d 883-908 566 5'-TTY GGI GGI GCI GGI GTI GGI
AAR AC 669.sup.d 445-470 814 5'-GCI GGC ACG TAC ACI GCC TG
666.sup.d 901-920 568 5'-RTI ATI GGI GCI GTI RTI GAY GT 669.sup.d
25-47 567.sup.c 5'-TCR TCI GCI GGI ACR TAI AYI GCY TG 669.sup.d
883-908 570 5'-RTI RYI GGI CCI GTI RTI GAY GT 672.sup.d 31-53
567.sup.c 5'-TCR TCI GCI GGI ACR TAI AYI GCY TG 669.sup.d 883-908
572 5'-RTI RTI GGI SCI GTI RTI GA 669.sup.d 25-44 567.sup.c 5'-TCR
TCI GCI GGI ACR TAI AYI GCY TG 669.sup.d 883-908 569 5'-RTI RTI GGI
SCI GTI RTI GAT AT 671.sup.d 31-53 567.sup.c 5'-TCR TCI GCI GGI ACR
TAI AYI GCY TG 669.sup.d 883-908 571 5'-RTI RTI GGI CCI GTI RTI GAT
GT 670.sup.d 31-53 567.sup.c 5'-TCR TCI GCI GGI ACR TAI AYI GCY TG
669.sup.d 883-908 .sup.a These sequences were aligned to derive the
corresponding primer. .sup.b The nucleotide positions refer to the
K. pneumoniae atpD sequence fragment (SEQ ID NO. 317). .sup.c These
sequences are from the complementary DNA strand of the sequence of
the originating fragment given in the Sequence Listing. .sup.d
Sequences from databases. Originating DNA fragment SEQ ID
Nucleotide SEQ ID NO. Nucleotide sequence NO. position Sequencing
primers (continued) 700 5'-TIR TIG AYG TCG ART TCC CTC ARG
669.sup.a 38-61 567.sup.b 5'-TCR TCI GCI GGI ACR TAI AYI GCY TG
669.sup.a 883-908 568 5'-RTI ATI GGI GCI GTI RTI GAY GT 669.sup.a
25-47 573.sup.b 5'-CCI CCI ACC ATR TAR AAI GC 666.sup.a 1465-1484
574 5'-ATI GCI ATG GAY GGI ACI GAR GG 666.sup.a 283-305 573.sup.b
5'-CCI CCI ACC ATR TAR AAI GC 666.sup.a 1465-1484 574 5'-ATI GCI
ATG GAY GGI ACI GAR GG 666.sup.a 283-305 708.sup.b 5'-TCR TCC ATI
CCI ARI ATI GCI ATI AT 666.sup.a 1258-1283 681 5'-GGI SSI TTY GGI
ISI GGI AAR AC 685 694-716 682.sup.b 5'-GTI ACI GGY TCY TCR AAR TTI
CCI CC 686 1177-1202 681 5'-GGI SSI TTY GGI ISI GGI AAR AC 685
694-716 683.sup.b 5'-GTI ACI GGI TCI SWI AWR TCI CCI CC 685
1180-1205
681 5'-GGI SSI TTY GGI ISI GGI AAR AC 685 694-716 699 5'-GTI ACI
GGY TCY TYR ARR TTI CCI CC 686 1177-1202 681 5'-GGI SSI TTY GGI ISI
GGI AAR AC 685 694-716 812.sup.b 5'-GTI ACI GGI TCY TYR ARR TTI CCI
CC 685 1180-1205 1213 5'-AAR GGI GGI ACI GCI GCI ATH CCI GG
714.sup.a 697-722 1212.sup.b 5'-CCI CCI RGI GGI GAI ACI GCW CC
714.sup.a 1189-1211 1203 5'-GGI GAR MGI GGI AAY GAR ATG 709.sup.a
724-744 1207.sup.b 5'-CCI TCI TCW CCI GGC ATY TC 709.sup.a 985-1004
1204 5'-GCI AAY AAC ITC IWM YAT GCC 709.sup.a 822-842 1206.sup.b
5'-CKI SRI GTI GAR TCI GCC A 709.sup.a 926-944 1205 5'-AAY ACI TCI
AWY ATG CCI GT 709.sup.a 826-845 1207.sup.b 5'-CCI TCI TCW CCI GGC
ATY TC 709.sup.a 985-1004 2282 5'-AGR RGC IMA RAT GTA TGA 714.sup.a
84-101 2284.sup.b 5'-TCT GWG TRA CIG GYT CKG AGA 714.sup.a
1217-1237 2283 5'-ATI TAT GAY GGK ITT CAG AGG C 714.sup.a 271-292
2285.sup.b 5'-CMC CIC CWG GTG GWG AWA C 714.sup.a 1195-1213
.sup.aSequences from databases. .sup.bThese sequences are from the
complementary DNA strand of the sequence of the originating
fragment given in the Sequence Listing.
TABLE-US-00042 Annex III: Internal hybridization probes for
specific detection of tuf sequences. Originating DNA fragment SEQ
ID Nucleotide SEQ ID NO. Nucleotide sequence NO. position Bacterial
species: Abiotrophia adiacens 2170 5'-ACG TGA CGT TGA CAA ACC A
1715 313-331 Bacterial species: Chlamydia pneumoniae 2089 5'-ATG
CTG AAC TTA TTG ACC TT 20 136-155 2090 5'-CGT TAC TGG AGT CGA AAT G
20 467-485 Bacterial species: Enterococcus faecalis 580 5'-GCT AAA
CCA GCT ACA ATC ACT CCA C 62-63, 607.sup.a 584-608.sup.b 603 5'-GGT
ATT AAA GAC GAA ACA TC 62-63, 607.sup.a 440-459.sup.b 1174 5'-GAA
CGT GGT GAA GTT CGC 62-63, 607.sup.a 398-415.sup.b Bacterial
species: Enterococcus faecium 602 5'-AAG TTG AAG TTG TTG GTA TT 64,
608.sup.a 426-445.sup.c Bacterial species: Enterococcus gallinarum
604 5'-GGT GAT GAA GTA GAA ATC GT 66, 609.sup.a 419-438.sup.d
Bacterial species: Escherichia coli 579 5'-GAA GGC CGT GCT GGT GAG
AA 78 503-522 2168 5'-CAT CAA AGT TGG TGA AGA AGT TG 78 409-431
Bacterial species: Neisseria gonorrhoeae 2166 5'-GAC AAA CCA TTC
CTG CTG 126 322-339.sup.e Fungal species: Candida albicans 577
5'-CAT GAT TGA ACC ATC CAC CA 407-411.sup.a 406-425.sup.f Fungal
species: Candida dubliniensis 578 5'-CAT GAT TGA AGC TTC CAC CA
412, 414-415.sup.a 418-437.sup.g .sup.aThese sequences were aligned
to derive the corresponding primer. .sup.bThe nucleotide positions
refer to the E. faecalis tuf sequence fragment (SEQ ID NO. 607).
.sup.cThe nucleotide positions refer to the E. faecium tuf sequence
fragment (SEQ ID NO. 608). .sup.dThe nucleotide positions refer to
the E. gallinarum tuf sequence fragment (SEQ ID NO. 609). .sup.eThe
nucleotide positions refer to the N. gonorrhoeae tuf sequence
fragment (SEQ ID NO. 126). .sup.fThe nucleotide positions refer to
the C. albicans tuf(EF-1) sequence fragment (SEQ ID NO. 408).
.sup.gThe nucleotide positions refer to the C. dubliniensis
tuf(EF-1) sequence fragment (SEQ ID NO. 414). Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Bacterial species: Haemophilus influenzae 581 5'-ACA TCG
GTG CAT TAT TAC GTG G 610.sup.a 551-572 Bacterial species:
Mycoplasma pneumoniae 2095 5'-CGG TCG GGT TGA ACG TGG 2097.sup.a
687-704 Bacterial species: Staphylococcus aureus 584 5'-ACA TGA CAC
ATC TAA AAC AA 176-180.sup.b 369-388.sup.c 585 5'-ACC ACA TAC TGA
ATT CAA AG 176-180.sup.b 525-544.sup.c 586 5'-CAG AAG TAT ACG TAT
TAT CA 176-180.sup.b 545-564.sup.c 587 5'-CGT ATT ATC AAA AGA CGA
AG 176-180.sup.b 555-574.sup.c 588 5'-TCT TCT CAA ACT ATC GTC CA
176-180.sup.b 593-612.sup.c Bacterial species: Staphylococcus
epidermidis 589 5'-GCA CGA AAC TTC TAA AAC AA 185, 611.sup.b
445-464.sup.d 590 5'-TAT ACG TAT TAT CTA AAG AT 185, 611.sup.b
627-646.sup.d 591 5'-TCC TGG TTC TAT TAC ACC AC 185, 611.sup.b
586-605.sup.d 592 5'-CAA AGC TGA AGT ATA CGT AT 185, 611.sup.b
616-635.sup.d 593 5'-TTC ACT AAC TAT CGC CCA CA 185, 611.sup.b
671-690.sup.d Bacterial species: Staphylococcus haemolyticus 594
5'-ATT GGT ATC CAT GAC ACT TC 186, 188-190.sup.b 437-456.sup.e 595
5'-TTA AAG CAG ACG TAT ACG TT 186, 188-190.sup.b 615-634.sup.e
Bacterial species: Staphylococcus hominis 596 5'-GAA ATT ATT GGT
ATC AAA GA 191, 193-196.sup.b 431-450.sup.f 597 5'-ATT GGT ATC AAA
GAA ACT TC 191, 193-196.sup.b 437-456.sup.f 598 5'-AAT TAC ACC TCA
CAC AAA AT 191, 193-196.sup.b 595-614.sup.f .sup.aSequences from
databases. .sup.bThese sequences were aligned to derive the
corresponding probe. .sup.cThe nucleotide positions refer to the S.
aureus tuf sequence fragment (SEQ ID NO. 179). .sup.dThe nucleotide
positions refer to the S. epidermidis tuf sequence fragment (SEQ ID
NO. 611). .sup.eThe nucleotide positions refer to the S.
haemolyticus tuf sequence fragment (SEQ ID NO. 186). .sup.fThe
nucleotide positions refer to the S. hominis tuf sequence fragment
(SEQ ID NO. 191). Originating DNA fragment SEQ ID Nucleotide SEQ ID
NO. Nucleotide sequence NO. position Bacterial species:
Staphylococcus saprophyticus 599 5'-CGG TGA AGA AAT CGA AAT CA
198-200.sup.a 406-425.sup.b 600 5'-ATG CAA GAA GAA TCA AGC AA
198-200.sup.a 431-450.sup.b 601 5'-GTT TCA CGT GAT GAT GTA CA
198-200.sup.a 536-555.sup.b 695 5'-GTT TCA CGT GAT GAC GTA CA
198-200.sup.a 563-582.sup.b Bacterial species: Streptococcus
agalactiae 582.sup.c 5'-TTT CAA CTT CGT CGT TGA CAC GAA CAG T
207-210.sup.a 404-431.sup.d 583.sup.c 5'-CAA CTG CTT TTT GGA TAT
CTT CTT TAA TAC CAA CG 207-210.sup.a 433-467.sup.d 1199 5'-GTA TTA
AAG AAG ATA TCC AAA AAG C 207-210.sup.a 438-462.sup.d Bacterial
species: Streptococcus pneumoniae 1201 5'-TCA AAG AAG AAA CTA AAA
AAG CTG T 971, 977, 513-537.sup.e 979, 986.sup.a Bacterial species:
Streptococcus pyogenes 1200 5'-TCA AAG AAG AAA CTA AAA AAG CTG T
1002 473-497 Bacterial group: Enterococcus
casseliflavus-flavescens- gallinarum group 620 5'-ATT GGT GCA TTG
CTA CGT 58, 65, 66.sup.a 527-544.sup.f 1122 5'-TGG TGC ATT GCT ACG
TGG 58, 65, 66.sup.a 529-546.sup.f Bacterial group: Enterococcus
sp., Gemella sp., A. adiacens 2172 5'-GTG TTG AAA TGT TCC GTA AA
58-62, 67-71, 477-496.sup.g 87-88, 607-609, 727, 871 1715,
1722.sup.a .sup.aThese sequences were aligned to derive the
corresponding primer. .sup.b The nucleotide positions refer to the
S. saprophyticus tuf sequence fragment (SEQ ID NO. 198).
.sup.cThese sequences are from the complementary DNA strand of the
sequence of the originating fragment given in the Sequence Listing.
.sup.dThe nucleotide positions refer to the S. agalactiae tuf
sequence fragment (SEQ ID NO. 209). .sup.eThe nucleotide positions
refer to the S. pneumoniae tuf sequence fragment (SEQ ID NO. 986).
.sup.fThe nucleotide positions refer to the E. flavescens tuf
sequence fragment (SEQ ID NO. 65). .sup.gThe nucleotide positions
refer to the E. faecium tuf sequence fragment (SEQ ID NO. 608).
Originating DNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide
sequence NO. position Bacterial genus: Gemella 2171 5'-TCG TTG GAT
TAA CTG AAG AA 87, 88.sup.a 430-449.sup.b Bacterial genus:
Staphylococcus sp. 605 5'-GAA ATG TTC CGT AAA TTA TT 176-203.sup.a
403-422.sup.c 606 5'-ATT AGA CTA CGC TGA AGC TG 176-203.sup.a
420-439.sup.c 1175 5'-GTT ACT GGT GTA GAA ATG TTC 176-203.sup.a
391-411.sup.c 1176 5'-TAC TGG TGT AGA AAT GTT C 176-203.sup.a
393-411.sup.c Bacterial genus: Streptococcus sp. 1202 5'-GTG TTG
AAA TGT TCC GTA AAC A 206-231, 971, 466-487.sup.d 977, 979,
982-986.sup.a Fungal species: Candida albicans 1156 5'-GTT GAA ATG
CAT CAC GAA CAA TT 407-412, 624.sup.a 680-702.sup.e Fungal group:
Candida albicans and C. tropicalis 1160 5'-CGT TTC TGT TAA AGA AAT
TAG AAG 407-412, 748-771.sup.e 429, 624.sup.a Fungal species:
Candida dubliniensis 1166 5'-ACG TTA AGA ATG TTT CTG TCA A
414-415.sup.a 750-771.sup.f 1168 5'-GAA CAA TTG GTT GAA GGT GT
414-415.sup.a 707-726.sup.f Fungal species: Candida glabrata 1158
5'-AAG AGG TAA TGT CTG TGG T 417 781-799 1159 5'-TGA AGG TTT GCC
AGG TGA 417 718-735 Fungal species: Candida krusei 1161 5'-TCC AGG
TGA TAA CGT TGG 422 720-737 .sup.aThese sequences were aligned to
derive the corresponding primer. .sup.bThe nucleotide positions
refer to the G. haemolysans tuf sequence fragment (SEQ ID NO. 87).
.sup.cThe nucleotide positions refer to the S. aureus tuf sequence
fragment (SEQ ID NO. 179). .sup.dThe nucleotide positions refer to
the S. pneumoniae tuf sequence fragment (SEQ ID NO. 986). .sup.eThe
nucleotide positions refer to the C. albicans tuf(EF-1) sequence
fragment (SEQ ID NO. 408). .sup.fThe nucleotide positions refer to
the C. dubliniensis tuf(EF-1) sequence fragment (SEQ ID NO. 414).
Originating DNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide
sequence NO. position Fungal group: Candida lusitaniae and C.
guillermondii 1162 5'-CAA GTC CGT GGA AAT GCA 418, 424.sup.a
682-699.sup.b Fungal species: Candida parapsilosis 1157 5'-AAG AAC
GTT TCA GTT AAG GAA AT 426 749-771 Fungal species: Candida
zeylanoides
1165 5'-GGT TTC AAC GTG AAG AAC 432 713-730 Fungal genus: Candida
sp. 1163 5'-GTT GGT TTC AAC GTT AAG AAC 407-412, 414-415,
728-748.sup.c 417, 418, 422, 429.sup.a 1164 5'-GGT TTC AAC GTC AAG
AAC 413, 416, 420, 740-757.sup.b 421, 424, 425, 426, 428, 431.sup.a
1167 5'-GTT GGT TTC AAC GT 406-426, 428-432, 728-741.sup.c
624.sup.a .sup.aThese sequences were aligned to derive the
corresponding primer. .sup.bThe nucleotide positions refer to the
C. lusitaniae tuf(EF-1) sequence fragment (SEQ ID NO. 424).
.sup.cThe nucleotide positions refer to the C. albicans tuf(EF-1)
sequence fragment (SEQ ID NO. 408).
TABLE-US-00043 Annex IV: Strategy for the selection of
amplification/sequencing primers from atpD (F-type) sequences. SEQ
ID 23 49 443 472 881 910 NO.: Accession #: B. cepacia AGTgCAT
CGGCGCCGTT ATCGACGTGG...TGTTCG GCGGTGCTGG CGTGGGCAAG ACCG...TCCA
GGCCGTGT ACGTCCCTGC GGACGACT -- X76877 B. pertussis AGTgCAT
CGGCGCCGTG GTGGATATTC...TGTTCG GCGGCGCCGG CGTGGGCAAG ACCG...TCCA
GGCCGTGT ACGTGCCTGC CGACGACT -- Genome project P. aeruginosa
AAATCAT CGGCGCCGTG ATCGACGTGG...TGTTCG GCGGCGCCGG CGTGGGCAAG
ACCG...TCCA GGCCGTAT ACGTTCCCGC GGACGACC -- Genome project E. coli
AGGTAAT CGGCGCCGTA GTTGACGTCG...TGTTCG GTGGTGCGGG TGTAGGTAAA
ACCG...TACA GGCAGTAT ACGTACCTGC GGATGACT -- J01594 N. gonorrhoeae
AAATTAT CGGTGCGGTT GTTGACGTGG...TGTTCG GCGGTGCCGG TGTGGGTAAA
ACCG...TCCA AGCCGTAT ATGTACCTGC GGATGACT -- Genome project M.
thermoacetica AGGTTAT TGGCCCGGTG GTTGACGTCG...TCTTCG GCGGCGCCGG
GGTCGGCAAG ACGG...TGCA AGCTATCT ATGTGCCGGC CGACGACC -- U64318 S.
aurantiaca AGGTTcT CGGTCCCGTG ATTGACGTGG...TGTTCG GCGGCGCCGG
CGTGGGCAAG ACGG...TGCA GGCCATCT ACGTGCCCGC CGACGACC -- X76879 M.
tuberculosis GGGTCAC TGGGCCCGTC GTCGACGTCG...TGTTCG GCGGTGCCGG
GGTGGGCAAG ACGG...TGCA AGCCGTCT ACGTGCCCGC CGACGACT -- Z73419 B.
fragilis AGGTAAT TGGCCCTGTG GTCGATGTGT...TGTTTG GCGGGGCCGG
AGTGGGTAAA ACTG...TGCA GGCTGTTT ACGTACCGGC TGATGACT -- M22247 C.
lytica AAATTAT TGGCCCAGTT ATAGATGTGG...TATTTG GAGGTGCCGG AGTAGGTAAA
ACAG...TACA GGCGGTTT ACGTACCTGC GGATGATT 672 M22535 A. woodii
AGGTTAT TGGACCAGTA GTCGATGTTA...TTTTCG GTGGTGCCGG AGTTGGTAAA
ACCG...TTCA GGCCGTTT ACGaTCCAGC CGATGACT -- U10505 C.
acetobutylicum AGGTAAT AGGACCTGTT GTGGATATTA...TGTTCG GTGGTGCCGG
TGTTGGTAAA ACAG...TTCA GGCTGTAT ATGTTCCTGC TGATGACC 671 AF101055 M.
pneumoniae AAGTGAT TGGCCCGGTA GTTGATGTCA...TATTTG GTGGTGCTGG
TGTTGGTAAA ACGG...TGCA AGCGATCT ATGTGCCAGC TGATGACT -- U43738 H.
pylori AGGTTtT AGGCCCGGTG GTAGATGTGG...TGTTTG GTGGGGCTGG CGTAGGCAAA
ACGG...TTCA AGCGGTGT ATGTGCCAGC AGACGACT 670 AF004014 Selected
sequences RTIAT IGGIGCIGTI RTIGAYGT 568 for universal primers RTIRY
IGGICCIGTI RTIGAYGT 570 RTIRT IGGISCIGTI RTIGA 572 RTIRT IGGISCIGTI
RTIGATAT 569 RTIRT IGGICCIGTI RTIGATGT 571 TTYG GIGGIGCIGG
IGTIGGIAAR AC 566 Selected sequence CA RGCIRTIT AYGTICCIGC IGAYGA
567 for universal primer.sup.a The sequence numbering refers to the
Escherichia coli atpD gene fragment (SEQ ID NO. 669). Nucleotides
in capitals are identical to the selected sequences or match those
sequences. Mismatches are indicated by lower-case letters. Dots
indicate gaps in the sequences displayed. "R" "Y" "M" "K" "W" and
"S" designate nucleotide positions which are degenerated. "R"
stands for A or G; "Y" stands for C or T; "M" stands for A or C;
"K" stands for G or T; "W" stands for A or T; "S" stands for C or
G. "I" stands for inosine which is a nucleotide analog that can
bind to any of the four nucleotides A, C, G or T. .sup.aThis
sequence is the reverse-complement of the selected primer.
TABLE-US-00044 Annex V: Strategy for the selection of universal
amplification/sequencing primers from atpD (V-type) sequences. SEQ
691 719 1177 1208 ID NO.: E. hirae CC AGGTCCGTTT GGTGCAGGGA
AGACAGT...TCTGGTGGAg ATaTCtctGA ACCAGTGACT CA 685 H. salinarum CC
GGGGCCGTTC GGGTCCGGGA AGACGGT...CCCGGCGGGg ACTTCtccGA GCCGGTCACC CA
687 T. thermophilus CC TGGGCCCTTC GGCAGCGGCA AGACCGT...CCGGGCGGCg
ACaTgtccGA GCCCGTGACC CA 693 Human CC TGGGGCCTTC GGATGTGGCA
AGACTGT...CCCGGTGGAg ACTTCtcAGA tCCCGTGACG AC 688 T. congolense CC
TGGCGCGTTT GGATGCGGAA AGACGGT...CCTGGAGGTg ACTTTtctGA cCCAGTGACG TC
692 P. falciparum CC TGGTGCATTT GGTTGTGGAA AAACTTG...CCAGGTGGTg
ATTTCtctGA cCCTGTAACT AC 689 C. pneumoniae CC AGGACCTTTT GGTGCAGGGA
AAACAGT...GCAGGAGGAA ACTTTGAAGA ACCAGTCACT CA 686 Selected
sequences GGISSITTY GGIISIGGIA ARAC 681 for universal primers
Selected sequences GGIGGIA AYTTYGARGA RCCIGTIAC 682 for universal
primers.sup.a GGIGGIG AYWTIWSIGA ICCIGTIAC 683 The sequence
numbering refers to the Enterococcus hirae atpD gene fragment (SEQ
ID NO. 685). Nucleotides in capitals are identical to the selected
sequences or match those sequences. Mismatches for SEQ ID NOs. 681
and 682 are indicated by lower-case letters. Mismatches for SEQ ID
NO. 683 are indicated by underlined nucleotides. Dots indicate gaps
in the sequences displayed. "R" "Y" "M" "K" "W" and "S" designate
nucleotide positions which are degenerated. "R" stands for A or G;
"Y" stands for C or T; "M" stands for A or C; "K" stands for G or
T; "W" stands for A or T; "S" stands for C or G. "I" stands for
inosine which is a nucleotide analog that can bind to any of the
four nucleotides A, C, G or T. .sup.aThese sequences are the
reverse-complement of the selected primers.
TABLE-US-00045 Annex VI: Strategy for the selection of universal
amplification/sequencing primers from tuf (M) sequences (organelle
origin). 601 635 C. neoformans.sup.a AAGAA CATGATCACC GGTaCCtCCC
AGgctGACTG... S. cerevisiae.sup.a AAGAA CATGATTACT GGTaCTtCTC
AAgctGACTG... O. volvulus.sup.a AAGAA TATGATCACA GGTaCTtCTC
AGgctGACTG... Human.sup.a AAAAA CATGATTACA GGGaCAtCTC AGgctGACTG...
G. maxB1.sup.b AAGAA CATGATCACC GGCGCTGCCC AGATGGACGG... G.
maxB2.sup.b AAAAA CATGATCACC GGCGCCGCCC AGATGGACGG... E. coli.sup.c
AAAAA CATGATCACC GGTGCTGCTC AGATGGACGG... S. aureofaciens.sup.c
AAGAA CATGATCACC GGTGCCGCCC AGATGGACGG... E. tenella.sup.b AAAAA
TATGATTACA GGAGCAGCAC AAATGGATGG... T. gondii.sup.b AAGAA
TATGATTACT GGAGCCGCAC AAATGGATGG... S. cerevisiae.sup.b AAGAA
TATGATTACC GGTGCTGCTC AAATGGATGG... A. thaliana.sup.b AAAAA
TATGATTACT GGAGCTGCGC AAATGGATGG... Selected sequence for AA
YATGATIACI GGIGCIGCIC ARATGGA universal primer Selected sequences
for universal primers SEQ ID Accession 1479 1511 NO.: #: C.
neoformans.sup.a CGCcgTCcGA GAcatGcGAC AGACcGTTGc CGT -- U81803 S.
cerevisiae.sup.a CGCTgTCAGA GAcatGaGAC AAACTGTcGc TGT 665 X00779 O.
volvulus.sup.a TGCTgTGcGt GAtatGaGAC AAACaGTTGc GGT -- M64333
Human.sup.a TGCTgTTcGt GAtatGaGAC AGACaGTTGc TGT -- X03558 G.
maxB1.sup.b TGCTATTAGA GAAGGAGGCA AAACTGTTGG AGC -- Y15107 G.
maxB2.sup.b TGCTATTAGA GAAGGAGGCA AAACTGTTGG AGC -- Y15108 E.
coli.sup.c CGCaATCcGt GAAGGGGCC GTACcGTTGG CGC 78 -- S.
aureofaciens.sup.c CGCcATCcGt GAGGGTGGTC GTACcGTgGG CGC -- AF007125
E. tenella.sup.b TGCTATAAGAGAAGGAGGAA AAACTATAGG AGC -- AI755521 T.
gondii.sup.b TGCTATTAGA GAAGGAGGTC GTACTATAGG AGC -- Y11431 S.
cerevisiae.sup.b CAATATCAGA GAGGGTGGAA GAACTGTTGG TAC 619 K00428 A.
thaliana.sup.b TGCctTAAGG GAAGGAGGTA GAACaGTTGG AGC -- X89227
Selected 664 sequence for universal primer Selected TATIAGR
GARGGIGGIM RIACTRTWGG.sup.d 652 sequences ATCCGT GAGGGYGGCC
GITCIGT.sup.d 561 for universal primers The sequence numbering
refers to the Saccharomyces cerevisiae tuf (M) gene (SEQ ID NO.
619). Nucleotides in capitals are identical to the selected
sequences or match those sequences. Mismatches for SEQ ID NOs. 652
and 664 are indicated by lower-case letters. Mismatches for SEQ ID
NO. 561 are indicated by underlined nucleotides. Dots indicate gaps
in the sequences displayed. "R" "Y" "M" "K" "W" and "S" designate
nucleotide positions which are degenerated. "R" stands for A or G;
"Y" stands for C or T; "M" stands for A or C; "K" stands for G or
T; "W" stands for A or T; "S" stands for C or G. "I" stands for
inosine which is a nucleotide analog that can bind to any of the
four nucleotides A, C, G or T. .sup.aThis sequence refers to
tuf(EF-1) gene. .sup.bThis sequence refers to tuf (M) or organelle
gene. .sup.cThis sequence refers to tuf gene from bacteria.
.sup.dThese sequences are the reverse-complement of the selected
primers.
TABLE-US-00046 Annex VII: Strategy for the selection of eukaryotic
sequencing primers from tuf (EF-1) sequences. SEQ ID Accession 154
179 286 314 NO.: #: S. cerevisiae GG TTCTTTCAAG TACGCTTGGG
TTTT...AGAGA TTTCATCAAG AACATGATTA CTGG... 665 X00779 B. hominis GG
CTCCTTCAAG TACGCGTGGG TGCT...CGTGA CTTCATaAAG AACATGATCA CGGG... --
D64080 C. albicans GG TTCTTTCAAA TACGCTTGGG TCTT...AGAGA TTTCATCAAG
AATATGATCA CTGG... -- M29934 C. neoformans TC TTCTTTCAAG TACGCTTGGG
TTCT...CGAGA CTTCATCAAG AACATGATCA CCGG... -- U81803 E. histolytica
GG ATCATTCAAA TATGCTTGGG TCTT...AGAGA TTTCATTAAG AACATGATTA CTGG...
-- M92073 G. lamblia GG CTCCTTCAAG TACGCGTGGG TCCT...CGCGA
CTTCATCAAG AACATGATCA CGGG... -- D14342 H. capsulatum AA ATCCTTCAAA
TATGCGTGGG TCCT...CGTGA CTTCATCAAG AACATGATCA CTGG... -- U14100
Human GG CTCCTTCAAG TATGCCTGGG TCTT...AGAGA CTTtATCAAA AACATGATTA
CAGG... -- X03558 L. braziliensis GC GTCCTTCAAG TACGCGTGGG
TGCT...CGCGA CTTCATCAAG AACATGATCA CCGG... -- U72244 O. volvulus GG
CTCATTTAAA TATGCTTGGG TATT...CGTGA TTTCATTAAG AATATGATCA CAGG... --
M64333 P. berghei GG TagTTTCAAA TATGCATGGG TTTT...AAAcA TTTtATTAAA
AATATGATTA CTGG... -- AJ224150 P. knowlesi GG AagTTTTAAG TACGCATGGG
TGTT...AAGGA TTTCATTAAA AATATGATTA CCGG... -- AJ224153 S. pombe GG
TTCCTTCAAG TACGCCTGGG TTTT...CGTGA TTTCATCAAG AACATGATTA CCGG... --
U42189 T. cruzi TC TTCTTTCAAG TACGCGTGGG TCTT...CGCGA CTTCATCAAG
AACATGATCA CGGG... -- L76077 Y. lipolytica GG TTCTTTCAAG TACGCTTGGG
TTCT...CGAGA TTTCATCAAG AACATGATCA CCGG... -- AF054510 Selected
sequences for TCITTYAAR TAYGCITGGG T 558 amplification primers GA
YTTCATYAAR AAYATGATYA C 560 GA YTTCATIAAR AAYATGAT 653 The sequence
numbering refers to the Saccharomyces cerevisiae tuf (EF-1) gene
fragment (SEQ ID NO. 665). Nucleotides in capitals are identical to
the selected sequences SEQ ID NOs. 558, 560 or 653, or match those
sequences. Mismatches for SEQ ID no. 558 and 560 are indicated by
lower-case letters. Mismatches for SEQ ID NO. 653 are indicated by
underlined nucleotides. Dots indicate gaps in the sequences
displayed. "R" "Y" "M" "K" "W" and "S" designate nucleotide
positions which are degenerated. "R" stands for A or G; "Y" stands
for C. or T; "M" stands for A or C; "K" stands for G or T; "W"
stands for A or T; "S" stands for C or G. "I" stands for inosine
which is a nucleotide analog that can bind to any of the four
nucleotides A, C, G or T. SEQ ID Accession 751 776 1276 1304 NO.:
#: S. cerevisiae ...GTTTACAA GATCGGTGGT ATTGGTAC...GACATG
AGACAAACTG TCGCTGTCGG TGT 665 X00779 B. hominis ...GTGTACAA
GATTGGCGGT ATTGGTAC...GATATG AGACAGACTG TCGCTGTCGG TAT -- D64080 C.
albicans ...GTTTACAA GATCGGTGGT ATTGGTAC...GATATG AGACAAACCG
TTGCTGTtGG TGT -- M29934 C. neoformans ...GTCTACAA GATCGGTGGT
ATCGGCAC...GACATG CGACAGACCG TTGCCGTtGG TGT -- U81803 E.
histolytica ...GTTTACAA GATTTcAGGT ATTGGAAC...GATATG AaACAAACCG
TTGCTGTtGG AGT -- M92073 G. lamblia ...GTCTACAA GATCTcGGGc
gTCGGGAC...~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~ -- D14342 H. capsulatum
...GTGTACAA AATCTcTGGT ATTGGCAC...GACATG AGACAAACCG TCGCTGTCGG TGT
-- U14100 Human ...GTCTACAA AATTGGTGGT ATTGGTAC...GATATG AGACAGACAG
TTGCgGTgGG TGT -- X03558 L. braziliensis ...GTGTACAA GATCGGCGGT
ATCGGCAC...GACATG CGCagAACGG TCGCCGTCGG CAT -- U72244 O. volvulus
...GTTTACAA AATTGGAGGT ATTGGAAC...GATATG AGACAAACAG TTGCTGTtGG CGT
-- M64333 P. berghei ...GTATACAA AATTGGTGGT ATTGGTAC...GATATG
AGACAAACAA TTGCTGTCGG TAT -- AJ224150 P. knowlesi ...GTATACAA
AATCGGTGGT ATTGGTAC...GATATG AGACAAACCA TTGCTGTCGG TAT -- AJ224153
S. pombe ...GTTTACAA GATCGGTGGT ATTGGTAC...GACATG CGTCAAACCG
TCGCTGTCGG TGT -- U42189 T. cruzi ...GTGTACAA GATCGGCGGT
ATCGGCAC...GACATG CGCCAGACGG TCGCCGTCGG CAT -- L76077 Y. lipolytica
...GTCTACAA GATCGGTGGT ATCGGCAC...GACATG CGACAGACCG TTGCTGTCGG TGT
-- AF054510 Selected sequence for TACAA RATYKGIGGT ATYGG 654
amplification primer Selected sequences for TACAA RATYKGIGGT ATYGG
655 amplification primers.sup.a ATG MGICARACIR TYGCYGTCGG 559 The
sequence numbering refers to the Saccharomyces cerevisiae tuf
(EF-1) gene fragment (SEQ ID NO. 665). Nucleotides in capitals are
identical to the selected sequences or match those sequences.
Mismatches are indicated by lower-case letters. "~" indicate
incomplete sequence data. Dots indicate gaps in the sequences
displayed. "R" "Y" "M" "K" "W" and "S" designate nucleotide
positions which are degenerated. "R" stands for A or G; "Y" stands
for C or T; "M" stands for A or C; "K" stands for G or T; "W"
stands for A or T; "S" stands for C or G. "I" stands for inosine
which is a nucleotide analog that can bind to any of the four
nucleotides A, C, G or T. .sup.aThis sequences are the
reverse-complement of the selected primers.
TABLE-US-00047 Annex VIII: Strategy for the selection of
Streptococcus agalactiae-specific amplification primers from tuf
sequences. SEQ Accession 305 334 517 542 ID NO.: #: S. agalactiae
CCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG
207 -- S. agalactiae CCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC
AACGTTGGTG TTCTTCTTCG TG 208 -- S. agalactiae CCAGAA CGTGATACTG
ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG 209 -- S.
agalactiae CCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG
TTCTTCTTCG TG 210 -- S. anginosus CCAGAA CGTGAcACTG ACAAACCaTT
gCTT...AGAt AACGTaGGgG TTCTTCTTCG TG 211 -- S. anginosus CCAGAA
CGTGATACTG ACAAACCaTT gCTT...AGAt AACGTaGGTG TTCTTCTTCG TG 221 --
S. bovis CCAaAA CGTGATACTG ACAAACCaTT gCTT...GGAt AACGTTGGTG
TTCTTCTTCG TG 212 -- S. gordonii CCAGAA CGTGAcACTG ACAAACCaTT
gCTT...AGAt AAtGTaGGTG TcCTTCTTCG TG 223 -- S. mutans CCAGAA
CGTGATACTG ACAAgCCgcT cCTT...GGAt AAtGTTGGTG TTCTcCTTCG TG 224 --
S. pneumoniae CCAGAA CGTGAcACTG ACAAACCaTT gCTT...AGAt AACGTaGGTG
TcCTTCTTCG TG 145.sup.a S. sanguinis CCAGAA CGcGATACTG ACAAgCCaTT
gCTT...GGAC AACGTaGGTG TgCTTCTcCG TG 227 -- S. sobrinus CCAaAA
CGcGATACTG AtAAgCCaTT gCTT...AGAt AACGTTGGTG TgCTTCTTCG TG 228 --
B. cepacia CCGGAg CGTGcagtTG ACggcgCgTT cCTG...CGAC AACGTTGGTa
TcCTgCTgCG cG 16 -- B. fragilis CCTccg CGcGATgtTG AtAAACCTTT
ctTG...TGAC AACGTaGGTc TgtTgCTTCG TG -- P33165 B. subtilis CCAGAA
CGcGAcACTG AaAAACCaTT caTG...TGAC AACaTTGGTG ccCTTCTTCG cG --
Z99104 C. diphtheriae CCAGAg CGTGAgACcG ACAAgCCaTT cCTC...CGAC
AACtgTGGTc TgCTTCTcCG TG 662 -- C. trachomatis CCAGAA aGaGAaAtTG
ACAAgCCTTT cTTA...AGAg AAtGTTGGat TgCTcCTcaG aG 22 -- E. coli
CCAGAg CGTGcgAtTG ACAAgCCgTT cCTg...TGAg AACGTaGGTG TTCTgCTgCG TG
78 -- G. vaginalis CCAact CacGATctTG ACAAgCCaTT cTTg...CGAt
RACacTGGTc TTCTTCTcCG cG 135.sup.a S. aureus CCAGAA CGTGATtCTG
ACAAACCaTT cATg...TGAC AACaTTGGTG catTatTaCG TG 179 -- Selected
sequence for GAA CGTGATACTG ACAAACCTTT A 549 species-specific
primer Selected sequence for C AACGTTGGTG TTCTTCTTC 550
species-specific primer.sup.b The sequence numbering refers to the
Streptococcus agalactiae tuf gene fragment (SEQ ID NO. 209).
Nucleotides in capitals are identical to the selected sequences or
match those sequences. Mismatches are indicated by lower-case
letters. Dots indicate gaps in the sequences displayed. "R" "Y" "M"
"K" "W" and "S" designate nucleotide positions which are
degenerated. "R" stands for A or G; "Y" stands for C or T; "M"
stands for A or C; "K" stands for G or T; "W" stands for A or T;
"S" stands for C or G. "I" stands for inosine which is a nucleotide
analog that can bind to any of the four nucleotides A, C, G or T.
.sup.aThe SEQ ID NO. refers to previous patent publication
WO98/20157. .sup.bThis sequence is the reverse-complement of the
selected primer.
TABLE-US-00048 Annex IX: Strategy for the selection of
Streptococcus agalactiae-specific hybridization probes from tuf
sequences. SEQ ID Accession 401 431 433 470 NO.: #: S. GGTACTGT
TaaaGTtAAt GACGAAGTTG AAATCGTTGG TATcAAAGAc GAaATCtctA AAGCAGTTGT
TA 206 acidominimus S. agalactiae GGTACTGT TCGTGTCAAC GACGAAGTTG
AAATCGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTGT TA 209 S. agalactiae
GGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA GATATCCAAA
AAGCAGTTGT TA 144.sup.a S. agalactiae GGTACTGT TCGTGTCAAC
GACGAAGTTG AAATCGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTGT TA 207 S.
agalactiae GGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA
GATATCCAAA AAGCAGTTGT TA 210 S. agalactiae GGTACTGT TCGTGTCAAC
GACGAAGTTG AAATCGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTGT TA 208 S.
anginosus GGTACTGT TaaaGTCAAC GACGAAGTTG AAATCGTTGG TATccgtGAt
GAaATCCAAA AAGCAGTTGT TA 211 S. anginosus GGTACTGT TaaaGTCAAC
GAtGAAGTTG AAATCGTTGG TATccgcGAg GAaATCCAAA AAGCAGTTGT TA 221 S.
bovis GGTACTGT TaaaGTCAAC GACGAAGTTG AAATCGTTGG TATccgtGAc
GAcATCCAAA AAGCtGTTGT TA 212 S. anginosus GGTACTGT TaaaGTCAAt
GAtGAAGTTG AAATCGTTGG TATTcgtGAc GAaATCCAAA AAGCAGTTGT TA 213 S.
cricetus GGTACTGT TaagGTCAAt GACGAAGTTG AAATCGTTGG TATcAAgGAc
GAaATCCAAA AAGCgGTTGT TA 214 S. cristatus GGTACTGT TCGTGTCAAC
GAtGAAaTcG AAATCGTTGG TATcAAAGAA GAaATCCAAA AAGCAGTTGT TA 215 S.
downei GGTACTGT TaagGTCAAC GACGAAGTTG AAATCGTTGG TATcAAgGAc
GAaATCCAAA AAGCAGTTGT TA 216 S. GGTACTGT TCGTGTCAAC GACGAAaTcG
AAATCGTTGG TATcAAAGAA GAaActaAAA AAGCtGTTGT TA 217 dysgalactiae S.
equi equi GGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TATcAgAGAc
GAgATCaAAA AAGCAGTTGT TA 218 S. ferus GGTACTGT aaGaGTCAAC
GAtGAAGTTG AAATCGTTGG TATcAAAGAc GAaATCactA AAGCAGTTGT TA 219 S.
gordonii GGTAtcGT TaaaGTCAAt GACGAAaTcG AAATCGTTGG TATcAAAGAA
GAaATCCAAA AAGCAGTTGT TA 220 S. macacae GGTACTGT TaagGTtAAt
GAtGAAGTTG AAATCGTTGG TATTcgtGAc GATATtCAAA AAGCAGTTGT TA 222 S.
gordonii GGTAtcGT TaaaGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA
GAaActCAAA AAGCAGTTGT TA 223 S. mutans GGTACTGT TaaaGTtAAC
GAtGAAGTTG AAATCGTTGG TATccgtGAt GAcATtCAAA AAGCtGTTGT TA 224 S.
oralis GGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA
GAaActCAAA AAGCAGTTGT TA -- P33170 S. GGTgtTGT TCGTGTCAAt
GAtGAAaTcG AAATCGTTGG TATcAAAGAA GAaATCCAAA AAGCAGTTGT TA 225
parasanguinis S. pneumoniae GGTAtcGT TaaaGTCAAC GACGAAaTcG
AAATCGTTGG TATcAAAGAA GAaActCAAA AAGCAGTTGT TA 145.sup.a S.
pyogenes GGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA
GAaActaAAA AAGCtGTTGT TA -- Genome project S. ratti GGTACTGT
TaaaGTCAAt GACGAAGTTG AAATCGTTGG TATccgtGAt GAcATCCAAA AAGCtGTTGT
TA 226 S. salivarius GGTgtTGT TCGTGTCAAt GACGAAGTTG AAATCGTTGG
TcTTAAAGAA GAcATCCAAA AAGCAGTTGT TA 146.sup.a S. sanguinis GGTAtcGT
TaaaGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaATCCAAA AAGCAGTTGT
TA 227 S. sobrinus GGTACTGT TaagGTCAAC GACGAAGTTG AAATCGTTGG
TATccgtGAc GATATCCAAA AAGCAGTTGT TA 228 S. suis GGTACTGT TCGTGTCAAC
GACGAAaTcG AAATCGTTGG TcTTcAAGAA GAaAaatcta AAGCAGTTGT TA 229 S.
uberis GGTACTGT TCGTGTCAAC GACGAAaTTG AAATCGTTGG TATcAAAGAA
GAaActaAAA AAGCAGTTGT TA 230 S. GGTgtTGT TCGTGTtAAt GACGAAGTTG
AAATCGTTGG TcTTAAAGAA GAaATCCAAA AAGCAGTTGT TA 231 vestibularis
Selected sequences for ACTGT TCGTGTCAAC GACGAAGTTG AAA 582
species-specific hybridization CGTTGG TATTAAAGAA GATATCCAAA AAAGTTG
583 probes.sup.b The sequence numbering refers to the Streptococcus
agalactiae tuf gene fragment (SEQ ID NO. 209). Nucleotides in
capitals are identical to the selected sequences or match those
sequences. Mismatches are indicated by lower-case letters. Dots
indicate gaps in the sequences displayed. .sup.aThe SEQ ID NO.
refers to previous patent publication WO98/20157. .sup.bThese
sequences are the reverse-complement of the selected probes.
TABLE-US-00049 Annex X: Strategy for the selection of Streptococcus
agalactiae-specific amplification primers from atpD sequences. SEQ
ID 39 80 203 234 368 399 NO.: S. agalactiae TT GATTGTCTAT
AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC
GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG 380 S. agalactiae TT
GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG
TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG 379 S.
agalactiae TT GATTGTCTAT AAAAATGGCG ATAAGTCACA
AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC GTTC...CTT ATTAGCACCT
TACTTAAAAG GTGGTAAAG 381 S. agalactiae TT GATTGTCTAT AAAAATGGCG
ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC GTTC...CTT
ATTAGCACCT TACTTAAAAG GTGGTAAAG 382 S. agalactiae TT GATTGTCTAT
AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC
GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG 383 S. bovis TT
GATTGTtTAT AAAgATGGCG ATAAGTCtCA AAAAaTcGTg...TAAaGAaA CTTTGGGTCG
TGTgTTtAAt GTTC...CcT tcTtGCcCCT TACcTAAAAG GTGGTAAAG --.sup.a S.
salivarius TT GgTcGTtTAT ActgATGaac AaAAGTCtaA
AcgtaTcGTg...TAAaGATA CccTtGGaCG TGTCTTtAAC GTTC...CTT gcTAGCcCCT
TACcTtAAgG GTGGTAAAG 387 S. pneumoniae cT tgTcGTCTAc AAAAATGaCG
AaAgaaaAac AAAAaTcGTc...TAAaGAaA CTTTGGGaCG TGTCTTCAAC GTTt...CcT
tcTtGCcCCT TACcTtAAAG GTGGTAAAG --.sup.b S. pyogenes TT GATTGTtTAT
AAAgATaGtG ATAAaaagCA AAAAaTcGTc...TAAaGAaA CTTTGGGaCG cGTCTTtAAt
GTaC...CcT tcTtGCcCCT TACcTtAAAG GTGGTAAAG --.sup.c S. anginosus cT
tgTaGTCTAT AAAAATGaCG AaAAtaaAtc AAAAaTcGTc...gAAaGAaA CacTtGGTCG
cGTCTTtAAC GTTt...CcT tTTAGCcCCc TACcTcAAAG GTGGgAAAG 386 S.
sanguinis cT tgTaGTCTAT AAAAATGatG AgAAaaaAtc AAAAaTcGTc...aAAGGAaA
CTcTaGGcCG gGT gTTCAAt GTTt...CcT gcTAGCACCT TAtcTgAAAG GTGGgAAAG
--.sup.d S. mutans TT GgTcGTtTAT AAAgATGGCG AcAAGTCtCA
AAgAaTtGTt...aAAaGAaA CacTaGGTCG TGTCTTtAAt GTTC...CcT tcTtGCcCCT
TAtcTtAAAG GTGGTAAAG --.sup.e B. anthracis gT aAaacagagc AAcgAaaaCG
gaAcaagcat tAActTAacA...TgAtGcaA CacTtGGTCG TGTaTTtAAC GTat...CTT
AcTtGCtCCT TACaTtAAgG GTGGTAAga 247 B. cereus gT aAaacaaagc
AAcgAaaaCG g...aagcat gAActTAacA...TgAtGcaA CacTtGGaCG TGTaTTCAAC
GTat...CTT AcTtGCtCCT TACaTtAAgG GTGGTAAga 248 E. faecium TT
agTTGTtTAT AAAAATGaCG AaAAtaaAtc AAAAGTtGTt...TAAaGAaA CaTTaGGTCG
cGTaTTCAAC GTaC...tTT gcTtGCcCCa TAtTTAAAAG GTGGgAAAG 292 E.
gallinarum TT GATcGTtTAc AAAAAaGaCG AgAAaaaAac
AAAAGTAGTA...aAcaGATA CTcTaGGcCG aGTaTTtAAt GTaC...tTT ATTAGCtCCT
TACTTAAAAG GTGGTAAAG 293 E. faecalis TT agTcGTtTAT AAAAATGGCG
AagcaaaACA AAAAGTAGTA...TAAaGATA CaTTaGGTCG TGTgTTtAAC GTTt...CTT
ATTAGCACCT TAtcTAAAAG GTGGTAAAG 291 E. coli Ta cgaTGctctT
gAggtgcaaa ATggtaatgA gcgtcTgGTg...TAAaGcgA CTcTGGGcCG TaTCaTgAAC
GTaC...CcT gaTgtgtCCg TtCgctAAgG GcGGTAAAG --.sup.f L.
monocytogenes Ta tAaatctgAT gcAgAaGaaG caccaaCtag
ccAAcTtact...TAcaGtaA CTcTtGGTCG TGTaTTtAAt GTat...CTT gcTAGCtCCT
TACTTAAAAG GTGGTAAAa 324 S. aureus gT tATTGatgtg cctAAaGaaG
AaggtaCAat AcAAcTAacA...TgAtGAaA CaTTaGGTCG TGTaTTtAAt GTaC...tTT
AcTAGCACCT TAtaTtAAAG GTGGTAAAa 366 S. epidermidis ca cATcGaagtT
cctAAaGaaG ATggagCgCt tcAAtTAacA...TgAcGtaA CTcTaGGaaG aGTgTTtAAC
GTaC...CTT ATTAGCACCT TACaTAAAAG GTGGTAAAa 370 Selected sequences
ATTGTCTAT AAAAATGGCG ATAAGTC 627 for species- AAAATGGCG ATAAGTCACA
AAAAGTA 628 specific primer Selected sequences GGATA CTTTGGGTCG
TGTCTTCAAC G 625 for species- ATTAGCACCT TACTTAAAAG GTGGTA 626
specific primers.sup.g The sequence numbering refers to the
Streptococcus agalactiae tuf gene fragment (SEQ ID NO. 380).
Nucleotides in capitals are identical to the selected sequences or
match those sequences. Mismatches are indicated by lower-case
letters. Dots indicate gaps in the sequences displayed.
.sup.a,d,e,fThese sequences were obtained from Genbank and have
accession #: a = AB009314, d = AF001955, e = U31170, and f =
V00311. .sup.b,cThese sequences were obtained from genome
sequencing projects, .sup.gThese sequences are the
reverse-complement of the selected primers.
TABLE-US-00050 Annex XI: Strategy for the selection of Candida
albicans/dubliniensis-specific amplification primers, Candida
albicans-specific hybridization probe and Candida
dubliniensis-specific hybridization probe from tuf sequences. SEQ
337 368 403 428 460 491 ID NO.: Accession #: C. albicans CGTC
AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C
AAATCCGGTA AAGTTACTGG TAAGACCTTG T 624 -- C. albicans CGTC
AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C
AAATCCGGTA AAGTTACTGG TAAGACCTTG T 409 -- C. albicans CGTC
AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C
AAATCCGGTA AAGTTACTGG TAAGACCTTG T 410 -- C. albicans CGTC
AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C
AAATCCGGTA AAGTTACTGG TAAGACCTTG T 407 -- C. albicans CGTC
AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C
AAATCCGGTA AAGTTACTGG TAAGACCTTG T 408 -- C. dubliniensis CGTC
AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtTC CACCAACT...C
AAATCCGGTA AgGTTACTGG TAAGACCTTG T 412 -- C. dubliniensis CGTC
AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtTC CACCAACT...C
AAATCCGGTA AgGTTACTGG TAAGACCTTG T 414 -- C. dubliniensis CGTC
AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtTC CACCAACT...C
AAATCCGGTA AgGTTACTGG TAAGACCTTG T 415 -- C. glabrata CATC
AAGAAGGTcG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCcaC CACCAACG...C
AAggCtGGTg tcGTcAagGG TAAGACCTTG T 417 -- C. guilliermondii CGTC
AAGAAGGTTG GTTACAACCC tAAGACTG...CAACATGA TTGAggCtTC tACCAACT...C
AAggCtGGTA AgtccACcGG TAAGACtTTG T 418 -- C. kefyr CATC AAGAAGGTcG
GTTACAACCC AAAGAATG...CAACATGA TTGAAgCcaC CACCAACG...C AAggCtGGTA
ccGTcAagGG TAAGACCTTG T 421 -- C. krusei CATC AAGAAGGTTG GTTACAACCC
AAAGACTG...CAACATGA TTGAAgCATC CACCAACT...C AAggCaGGTg ttGTTAagGG
TAAGACCTTA T 422 -- C. lusitaniae CGTC AAGAAGGTTG GTTACAACCC
tAAGACTG...CAACATGA TTGAgCCATC YACCAACT...C AAgTCYGGTA AgtccACcGG
TAAGACCTTG T 424 -- C. neoformans CATC AAGAAGGTTG GTTACAACCC
cAAGgCTG...CAACATGt TgGAggagaC CACCAAGT...C AAgTCtGGTg tttccAagGG
TAAGACCcTC C 623 -- C. parapsilosis CGTC AAGAAGGTTG GTTACAACCC
tAAagCTG...CAAtATGA TTGAACCATC aACCAACT...T AAAgCtGGTA AgGTTACcGG
TAAGACCTTG T 426 -- C. tropicalis CGTC AAGAAGGTTG GTTACAACCC
tAAGgCTG...CAACATGA TTGAAgCtTC tACCAACT...C AAggCtGGTA AgGTTACcGG
TAAGACtTTG T 429 -- A. fumigatus CATC AAGAAGGTcG GcTACAACCC
cAAGgCCG...CAACATGc TTGAgCCcTC CtCCAACT...C AAggCCGGcA AgGTcACTGG
TAAGACCcTC A 404 -- Human CATt AAGAAaaTTG GcTACAACCC
cgAcACAG...CAACATGc TgGAgCCAag tgCtAACA...T AAggatGGcA AtGccAgTGG
aAccACgcTG C -- X03558 P. anomala TATC AAGAAaGTTG GTTACAACCC
AAAaACTG...TAACATGA TTGAACCATC aWCtAACT...C AAAgCtGGTg AAGcTAaaGG
TAAaACtTTA T 447 -- S. cerevisiae TATC AAGAAGGTTG GTTACAACCC
AAAGACTG...CAACATGA TTGAAgCtaC CACCAACG...C AAggCCGGTg tcGTcAagGG
TAAGACtTTG T 622 -- S. pombe CATC AAGAAGGTcG GTTtCAACCC
cAAGACCG...TAACATGA TTGAgCCcaC CACCAACA...C AAggCtGGTg tcGTcAagGG
TAAGACtcTT T -- U42189 Selected sequence C AAGAAGGTTG GTTACAACCC
AAAGA for species-specific amplification primer.sup.a Selected
sequence ATCCGGTA AAGTTACTGG TAAGACCT for species-specific
amplification primer.sup.a,b Selected sequences CATGA TTGAACCATC
CACCA (C. albicans) 577 for species-specific CATGA TTGAAGCTTC CACCA
(C. dubliniensis) 578 hybridization probes The sequence numbering
refers to the Candida albicans tuf gene fragment (SEQ ID NO. 408).
Nucleotides in capitals are identical to the selected sequences or
match those sequences. Mismatches for SEQ ID NO. 577 are indicated
by lower-case letters. Mismatches for SEQ ID NO. 578 are indicated
by underlined nucleotides. Dots indicate gaps in the sequerices
displayed. "R" "Y" "M" "K" "W" and "S" designate nucleotide
positions which are degenerated. "R" stands for A or G; "Y" stands
for C or T; "M" stands for A or C; "K" stands for G or T; "W"
stands for A or T; "S" stands for C or G. "I" stands for inosine
which is a nucleotide analog that can bind to any of the four
nucleotides A, C, G or T. .sup.aC. albicans primers have been
described in a previous patent (publication WO98/20157, SEQ ID NOs.
11-12) .sup.bThis sequence is the reverse-complement of the
selected primer.
TABLE-US-00051 Annex XII: Strategy for the selection of
Staphylococcus-specific amplification primers from tuf sequences.
SEQ ID Accession 310 340 652 682 NO.: #: S. aureus A CAGGCCGTGT
TGAACGTGGT CAAATCAAAG...CACTTACCA GAAGGTACTG AAATGGTAAT GC 179 --
S. aureus A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACC~ ~~~~~~~~~~
~~~~~~~~~~ GC 176 -- S. aureus A CAGGCCGTGT TGAACGTGGT
CAAATCAAAG...CACTTACCA GAAGGTMCTG AAATGGTAAT GC 177 -- S. aureus
aureus A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACCA GAAGGTACTG
AAATGGTAAT GC 180 -- S. auricularis A CAGGCCGTGT TGAACGTGGT
CAAATCAAAG...ActTTACCA GAAGGTACaG AAATGGTAAT GC 181 -- S. capitis A
CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTTAT
GC 182 -- capitis M. caseolyticus A CTGGaCGTGT TGAgCGTGGa
CAAgTtAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT GC 183 -- S. cohnii A
CAGGgCGTGT TGAACGTGGT CAAATCAAAG...ActTTACCA GAAGGTACTG AAATGGTTAT
GC 184 S. epidermidis A CAGGCCGTGT TGAACGTGGT
CAAATCAAAG...~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~ 185 -- S.
epidermidis A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA
GAAGGTACaG AAATGGTTAT GC 141.sup.a -- S. haemolyticus A CAGGCCGTGT
TGAACGTGGg CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTTAT GC 186 --
S. haemolyticus A CAGGtCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA
GAAG~~~~~~ ~~~~~~~~~~ ~~ 188 -- S. haemolyticus A CAGGCCGTGT
TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGG~~~~ ~~ 189 --
S. hominis A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA
GAAGGTACTG AAATGGTAAT GC 191 -- hominis S. hominis A CAGGCCGTGT
TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT GC 193 --
S. hominis A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA
GAAGG~~~~~ ~~~~~~~~~~ ~~ 194 -- S. hominis A CAGGCCGTGT TGAACGTGGT
CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT GC 195 -- S. hominis A
CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT
GC 196 -- S. lugdunensis A CAGGCCGTGT TGAACGTGGT
CAAATCAAAG...AACTTACCA GAAGGTACaG AAATGGTTAT GC 197 -- S.
saprophyticus A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...~~~~~~~~~
~~~~~~~~~~ ~~~~~~~~~~ ~~ 198 -- S. saprophyticus A CAGGCCGTGT
TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTTAT GC 199 --
S. saprophyticus A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA
GAAGGTACTG AAATGGTTAT GC 200 -- S. sciuri sciuri A CAGGCCGTGT
TGAACGTGGT CAAATCACTG...AACTTACCA GAAGGTACTG AAATGGTTAT GC 201 --
S. warneri A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CAaTTACCA
GAAGGTACTG ~~~~~~~~~~ ~~ 187 -- S. warneri A CAGGCCGTGT TGAACGTGGT
CAAATCAAAG...~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~ 192 -- S. warneri A
CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CAaTTACCA GAAGGTACTG AAATGGTTAT
GC 202 -- B. subtilis A CTGGCCGTGT aGAACGcGGa
CAAgTtAAAG...CAtcTtCCA GAAGGcgtaG AAATGGTTAT GC -- Z99104 E. coli A
CCGGtCGTGT aGAACGcGGT atcATCAAAG...GAacTgCCg GAAGGcgtaG AgATGGTAAT
GC 78 -- L. monocytogenes A CTGGaCGTGT TGAACGTGGa
CAAgTtAAAG...AcacTtCCA GAAGGTACTG AAATGGTAAY GC 138.sup.a --
Selected GGCCGTGT TGAACGTGGT CAAATCA 553 sequence for genus-
specific primer Selected TTACCA GAAGGTACTG AAATGGTIA 575 sequences
TTACCA GAAGGTACTG AAATGCTWA 707 for genus- specific primers.sup.b
The sequence numbering refers to the Staphylococcus aureus tuf gene
fragment (SEQ ID NO. 179). Nucleotides in capitals are identical to
the selected sequences or match those sequences. Mismatches are
indicated by lower-case letters. "~" indicate incomplete sequence
data. Dots indicate gaps in the sequences displayed. "R" "Y" "M"
"K" "W" and "S" designate nucleotide positions which are
degenerated. "R" stands for A or G; "Y" stands for C or T; "M"
stands for A or C; "K" stands for G or T; "W" stands for A or T;
"S" stands for C or G. "I" stands for inosine which is a nucleotide
analog that can bind to any of the four nucleotides A, C, G or T.
.sup.aThe SEQ ID NO. refers to previous patent publication
WO98/20157. .sup.bThese sequences are the reverse-complement of the
selected primers.
TABLE-US-00052 Annex XIII: Strategy for the selection of the
Staphylococcus-specific hybridization probe from tuf sequences. 400
425 SEQ ID NO.: Accession #: S. aureus G TTGAAATGTT CCGTAAATTA
TTAGA 179 -- S. aureus G TTGAAATGTT CCGTAAATTA TTAGA 176 -- S.
aureus G TTGAAATGTT CCGTAAATTA TTAGA 177 -- S. aureus G TTGAAATGTT
CCGTAAATTA TTAGA 178 -- S. aureus aureus G TTGAAATGTT CCGTAAATTA
TTAGA 180 -- S. auricularis G TAGAAATGTT CCGTAAATTA TTAGA 181 -- S.
capitis capitis G TAGAAATGTT CCGTAAATTA TTAGA 182 -- M.
caseolyticus G TAGAAATGTT CCGTAAATTA TTAGA 183 -- S. cohnii G
TAGAAATGTT CCGTAAATTA TTAGA 184 -- S. epidermidis G TAGAAATGTT
CCGTAAATTA TTAGA 185 -- S. haemolyticus G TAGAAATGTT CCGTAAATTA
TTAGA 186 -- S. haemolyticus G TAGAAATGTT CCGTAAATTA TTAGA 189 --
S. haemolyticus G TAGAAATGTT CCGTAAATTA TTAGA 190 -- S.
haemolyticus G TAGAAATGTT CCGTAAATTA TTAGA 188 -- S. hominis G
TAGAAATGTT CCGTAAATTA TTAGA 196 -- S. hominis G TAGAAATGTT
CCGTAAATTA TTAGA 194 -- S. hominis hominis G TAGAAATGTT CCGTAAATTA
TTAGA 191 -- S. hominis G TAGAAATGTT CCGTAAATTA TTAGA 193 -- S.
hominis G TAGAAATGTT CCGTAAATTA TTAGA 195 -- S. lugdunensis G
TAGAAATGTT CCGTAAATTA TTAGA 197 -- S. saprophyticus G TAGAAATGTT
CCGTAAATTA TTAGA 198 -- S. saprophyticus G TAGAAATGTT CCGTAAATTA
TTAGA 200 -- S. saprophyticus G TAGAAATGTT CCGTAAATTA TTAGA 199 --
S. sciuri sciuri G TTGAAATGTT CCGTAAATTA TTAGA 201 -- S. warneri G
TAGAAATGTT CCGTAAgTTA TTAGA 187 -- S. warneri G TAGAAATGTT
CCGTAAgTTA TTAGA 192 -- S. warneri G TAGAAATGTT CCGTAAgTTA TTAGA
202 -- S. warneri G TAGAAATGTT CCGTAAgTTA TTAGA 203 -- B. subtilis
G TTGAAATGTT CCGTAAgcTt cTTGA -- Z99104 E. coli G TTGAAATGTT
CCGcAAAcTg cTGGA 78 -- L. monocytogenes G TAGAAATGTT CCGTAAATTA
cTAGA .sup. 138.sup.a -- Selected sequence for GAAATGTT CCGTAAATTA
TT 605 genus-specific hybridization probe The sequence numbering
refers to the Staphylococcus aureus tuf gene fragment (SEQ ID NO.
179). Nucleotides in capitals are identical to the selected
sequence or match that sequence. Mismatches are indicated by
lower-case letters. .sup.aThe SEQ ID NO. refers to previous patent
publication WO98/20157.
TABLE-US-00053 Annex XIV: Strategy for the selection of
Staphylococcus saprophyticus-specific and of Staphylococcus
haemolyticus-specific hybridization probes from tuf sequences. SEQ
ID 339 383 NO.: S. aureus AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC
ATGACACaTC TAA 179 S. aureus AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC
ATGACACaTC TAA 176 S. aureus AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC
ATGACACaTC TAA 177 S. aureus AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC
ATGACACaTC TAA 178 S. aureus aureus AG TtGGTGAAGA AgTtGAAATC
ATcGGTtTaC ATGACACaTC TAA 180 S. auricularis AG TCGGTGAAGA
AgTtGAAATC ATcGGTATga AaGACggTTC AAA 181 S. capitis capitis AG
TtGGTGAAGA AgTtGAAATC ATcGGTATCC AcGAaACTTC TAA 182 M. caseolyticus
AG TtGGTGAAGA AgTtGAAATC ATTGGTtTaa cTGAagaacC AAA 183 S. cohnii AG
TCGGTGAAGA AgTtGAAATC ATcGGTATgC AaGAagaTTC CAA 184 S. epidermidis
AG TtGGTGAAGA AgTtGAAATC ATcGGTATgC AcGAaACTTC TAA 185 S.
haemolyticus AG TtGGTGAAGA AgTtGAAATC ATTGGTATCC ATGACACTTC TAA 186
S. haemolyticus AG TtGGTGAAGA AgTtGAAATC ATTGGTATCC ATGACACTTC TAA
189 S. haemolyticus AG TtGGTGAAGA AgTtGAAATC ATTGGTATCC ATGACACTTC
TAA 190 S. haemolyticus AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa
AaGAaACTTC TAA 188 S. hominis AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa
AaGAaACTTC TAA 194 S. hominis hominis AG TtGGTGAAGA AgTtGAAATt
ATTGGTATCa AaGAaACTTC TAA 191 S. hominis AG TtGGTGAAGA AgTtGAAATt
ATTGGTATCa AaGAaACTTC TAA 193 S. hominis AG TtGGTGAAGA AgTtGAAATt
ATTGGTATCa AaGAaACTTC TAA 195 S. hominis AG TtGGTGAAGA AgTtGAAATt
ATTGGTATCa AaGAtACTTC TAA 196 S. lugdunensis AG TCGGTGAAGA
AgTtGAAATt ATTGGTATCC AcGAtACTaC TAA 197 S. saprophyticus AG
TCGGTGAAGA AATCGAAATC ATcGGTATgC AaGAagaaTC CAA 198 S.
saprophyticus AG TCGGTGAAGA AATCGAAATC ATcGGTATgC AaGAagaaTC CAA
200 S. saprophyticus AG TCGGTGAAGA AATCGAAATC ATcGGTATgC AaGAagaaTC
CAA 199 S. sciuri sciuri TG TtGGTGAAGA AgTtGAAATC ATcGGTtTaa
cTGAagaaTC TAA 201 S. warneri AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC
ATGACACTTC TAA 187 S. warneri AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC
ATGACACTTC TAA 192 S. warneri AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC
ATGACACTTC TAA 202 S. warneri AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC
ATGACACTTC TAA 203 B. subtilis AG TCGGTGAcGA AgTtGAAATC ATcGGTcTtC
AaGAagagag AAA --.sup.a E. coli AG TtGGTGAAGA AgTtGAAATC gTTGGTATCa
AaGAgACTca GAA 78 L. monocytogenes AG TtGGTGAcGA AgTaGAAgTt
ATcGGTATCg AaGAagaaag AAA 138.sup.b Selected sequences for
CGGTGAAGA AATCGAAATC A (S. saprophyticus) 599 species-specific (S.
haemolyticus) ATTGGTATCC ATGACACTTC 594 hybridization probes The
sequence numbering refers to the Staphylococcus aureus tuf gene
fragment (SEQ ID NO. 179). Nucleotides in capitals are identical to
the selected sequences or match those sequences. Mismatches are
indicated by lower-case letters. .sup.aThis sequence was obtained
from Genbank accession #Z99104. .sup.bThe SEQ ID NO. refers to
previous patent publication WO98/20157.
TABLE-US-00054 Annex XV: Strategy for the selection of
Staphylococcus aureus-specific and of Staphylococcus
epidermidis-specific hybridization probes from tuf sequences. SEQ
ID 521 547 592 617 NO.: S. aureus TACACCACA TACTGAATTC
AAAGCAG...TTCTTCtCa AACTA TCGtC CACAAAT 179 S. aureus TACACCACA
TACTGAATTC AAAGCAG...TTCTTCtC~ ~~~~~~~~~~ ~~~~~~~ 178 S. aureus
TACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTA TCGtC CACAAAT 176 S.
aureus TACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTA TCGtC CACAAAT
177 S. aureus aureus TACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTA
TCGtC CACAAAT 180 S. auricularis TACACCACA cACTaAATTC
ActGCAG...TTCTTCtCT AACTA cCGtC CACAAAT 181 S. capitis capitis
CACACCACA cACTaAATTC AAAGCGG...TTCTTCAgT AACTA cCGCC CACAAAT 182 M.
caseolyticus TACtCCACA TACTaAATTC AAAGCTG...TTCTTCACT AACTA cCGCC
CtCAGTT 183 S. cohnii TACACCACA cACaaAcTTt AAAGCGG...TTCTTCAgT
AACTA TCGCC CACAAAT 184 S. epidermidis TACACCACA cACaaAATTC
AAAGCTG...TTCTTCACT AACTA TCGCC CACAAAT 185 S. haemolyticus
CACACCtCA cACaaAATTt AAAGCAG...TTCTTCACa AACTA TCGtC CACAAAT 186 S.
haemolyticus CACACCtCA cACaaAATTt AAAGCAG...TTCTTCACa AACTA TCGtC
CACAAAT 189 S. haemolyticus CACACCtCA cACaaAATTt
AAAGCAG...TTCTTCACa AACTA TCGtC CACAAAT 190 S. haemolyticus
TACACCtCA cACaaAATTC AAAGCAG...TTCTTCACT AACTA TCGtC CACAAAT 188 S.
hominis CACACCtCA cACaaAATTC AAAGCAG...TTCTTCACT AACTA TCGtC
CACAAAT 195 S. hominis TACACCtCA cACaaAATTC AAAGCAG...TTCTTCACT
AACTA TCGtC CACAAAT 196 S. hominis hominis TACACCtCA cACaaAATTC
AAAGCAG...TTCTTCtCT AACTA TCGtC CACAAAT 191 S. hominis TACACCtCA
cACaaAATTC AAAGCAG...TTCTTCtCT AACTA TCGtC CACAAAT 193 S. hominis
TACACCtCA cACaaAATTC AAAGCAG...TTCTTCtCT AACTA TCGtC CACAAAT 194 S.
lugdunensis TACACCtCA cACTaAATTt AAAGCTG...TTCTTCtCa AACTA cCGCC
CACAAAT 197 S. saprophyticus TACACCACA TACaaAATTC
AAAGCGG...TTCTTCACT AACTA cCGCC CACAAAT 198 S. saprophyticus
TACACCACA TACaaAATTC AAAGCGG...TTCTTCACT AACTA cCGCC CACAAAT 199 S.
saprophyticus TACACCACA TACaaAATTC AAAGCGG...TTCTTCACT AACTA cCGCC
CACAAAT 200 S. sciuri sciuri CACACCtCA cACTaAATTC
AAAGCTG...TTCTTCACa AACTA cCGCC CACAAAT 201 S. warneri TACACCACA
TACaaAATTC AAAGCGG...~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~ 192 S. warneri
TACACCACA TACaaAATTC AAAGCGG...TTCTTCAgT AACTA cCGCC CACAAAT 187 S.
warneri TACACCACA TACaaAATTC AAAGCGG...TTCTTCAgT AACTA cCGCC
CACAAAT 202 S. warneri TACACCACA TACaaAATTC AAAGCGG...TTCTTCAgT
AACTA cCGCC CACAAAT 203 B. subtilis CACtCCACA cAgcaAATTC
AAAGCTG...TTCTTCtCT AACTA cCGtC CtCAGTT --.sup.a E. coli CAAgCCgCA
cACcaAgTTC gAAtCTG...TTCTTCAaa ggCTA cCGtC CgCAGTT 78 L.
monocytogenes TACtCCACA cACTaAcTTC AAAGCTG...TTCTTCAac AACTA cCGCC
CACAAAT 138.sup.b Selected sequences ACCACA TACTGAATTC AAAG (S.
aureus) 585 for species-specific (S. epidermidis) TTCACT AACTATCGCC
CACA 593 hybridization probes The sequence numbering refers to the
Staphylococcus aureus tut gene fragment (SEQ ID NO. 179).
Nucleotides in capitals are identical to the selected sequences or
match those sequences. Mismatches are indicated by lower-case
letters. "~" indicate incomplete sequence data. Dots indicate gaps
in the sequences displayed. .sup.aThis sequence was obtained from
Genbank accession #Z99104. .sup.bThe SEQ ID NO. refers to previous
patent publication WO98/20157.
TABLE-US-00055 Annex XVI: Strategy for the selection of the
Staphylococcus hominis-specific hybridization probe from tuf
sequences. 358 383 SEQ ID NO.: S. aureus ATC ATcGGTtTac AtGAcACaTC
TAA 179 S. aureus ATC ATcGGTtTac AtGAcACaTC TAA 176 S. aureus ATC
ATcGGTtTac AtGAcACaTC TAA 177 S. aureus ATC ATcGGTtTac AtGAcACaTC
TAA 178 S. aureus aureus ATC ATcGGTtTac AtGAcACaTC TAA 180 S.
auricularis ATC ATcGGTATgA AAGAcggTTC AAA 181 S. capitis capitis
ATC ATcGGTATCc AcGAAACTTC TAA 182 M. caseolyticus ATC ATTGGTtTaA
ctGAAgaacC AAA 183 S. cohnii ATC ATcGGTATgc AAGAAgaTTC CAA 184 S.
epidermidis ATC ATcGGTATgc AcGAAACTTC TAA 185 S. haemolyticus ATC
ATTGGTATCc AtGAcACTTC TAA 186 S. haemolyticus ATC ATTGGTATCc
AtGAcACTTC TAA 189 S. haemolyticus ATC ATTGGTATCc AtGAcACTTC TAA
190 S. haemolyticus ATT ATTGGTATCA AAGAAACTTC TAA 188 S. hominis
ATT ATTGGTATCA AAGAtACTTC TAA 196 S. hominis ATT ATTGGTATCA
AAGAAACTTC TAA 194 S. hominis hominis ATT ATTGGTATCA AAGAAACTTC TAA
191 S. hominis ATT ATTGGTATCA AAGAAACTTC TAA 193 S. hominis ATT
ATTGGTATCA AAGAAACTTC TAA 195 S. lugdunensis ATT ATTGGTATCc
AcGAtACTaC TAA 197 S. saprophyticus ATC ATcGGTATgc AAGAAgaaTC CAA
198 S. saprophyticus ATC ATcGGTATgc AAGAAgaaTC CAA 200 S.
saprophyticus ATC ATcGGTATgc AAGAAgaaTC CAA 199 S. sciuri sciuri
ATC ATcGGTtTaA ctGAAgaaTC TAA 201 S. warneri ATC ATcGGTtTac
AtGAcACTTC TAA 187 S. warneri ATC ATcGGTtTac AtGAcACTTC TAA 192 S.
warneri ATC ATcGGTtTac AtGAcACTTC TAA 202 S. warneri ATC ATcGGTtTac
AtGAcACTTC TAA 203 B. subtilis ATC ATcGGTcTtc AAGAAgagag AAA
--.sup.a E. coli ATC gTTGGTATCA AAGAgACTca GAA 78 L. monocytogenes
GTT ATcGGTATCg AAGAAgaaag AAA 138.sup.b Selected sequence for
ATTGGTATCA AAGAAACTTC 597 species-specific hybridization probe The
sequence numbering refers to the Staphylococcus aureus tuf gene
fragment (SEQ ID NO. 179). Nucleotides in capitals are identical to
the selected sequences or match those sequences. Mismatches are
indicated by lower-case letters. Dots indicate gaps in the
sequences displayed. .sup.aThis sequence was obtained from Genbank
accession #Z99104. .sup.bThe SEQ ID NO. refers to previous patent
publication WO98/20157.
TABLE-US-00056 Annex XVII: Strategy for the selection of the
Enterococcus-specific amplification primers from tuf sequences. SEQ
ID Accession 270 298 556 582 NO.: #: E. avium TAGAATTAAT GGCTGCTGTT
GACGAATAT...TGAA GATATCCAAC GTGGACAAGTATT 131.sup.a -- E.
casseliflavus TGGAATTAAT GGCTGCAGTT GACGAATAC...TGAA GACATCCAAC
GTGGACAAGT ATT 58 -- E. cecorum TAGAATTAAT GGCTGCAGTT
GACGAATAC...TGAA GATATCCAAC GTGGtCAAGT ATT 59 -- E. dispar
TAGAATTAAT GGCTGCAGTT GACGAATAT...TGAA GATATCCAAC GTGGtCAAGT ATT 60
-- E. durans TTGAATTAAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC
GTGGACAAGT TTT 61 -- E. flavescens TGGAATTAAT GGCTGCAGTT
GACGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT 65 -- E. faecium
TTGAATTAAT GGCTGCAGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT TTT
608 -- E. faecalis TAGAATTAAT GGCTGCAGTT GACGAATAT...TGAA
GATATCGAAC GTGGACAAGT ATT 607 -- E. gallinarum TGGAATTgAT
GGCTGCAGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT 609 -- E.
hirae TTGAATTgAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC GTGGACAAGT
TTT 67 -- E. mundtii TTGAATTgAT GGCTGCAGTT GACGAATAT...TGAA
GACATCCAAC GTGGtCAAGT TTT 68 -- E. pseudoavium TAGAATTAAT
GSCTGCTGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT 69 -- E.
raffinosus TAGAATTAAT GGCTGCTGTT GATGAATAC...TGAA GACATCCAAC
GTGGACAAGT ATT 70 -- E. saccharolyticus TCGAATTAAT GGCTGCAGTT
GACGAATAT...TGAA GACATCCAAC GTGGACAAGT ATT 71 -- E. solitarius
TGGAcTTAAT GGaTGCAGTT GATGAcTAC...TGAt GATATCGAAC GTGGtCAAGT ATT 72
-- E. coli TGGAAcTggc tGgcttccTg GATtctTAY...TGAA GAaATCGAAC
GTGGtCAgGT ACT 78 -- B. cepacia TGAgccTggc cGacGCgcTg
GACacgTAC...TGAA GACgTgGAgC GTGGcCAgGT TCT 16 -- B. fragilis
TGGAAcTgAT GGaaGCTGTT GATactTGG...GAAc GAaATCaAAC GTGGtatgGT TCT --
M22247 B. subtilis TCGAAcTtAT GGaTGCgGTT GATGAgTAC...TGAA
GAaATCCAAC GTGGtCAAGT ACT -- Z99104 C. diphtheriae TCGAccTcAT
GcagGCTtgc KATGAtTCC...CGAA GACgTtGAgC GTGGcCAgGT TGT 662 -- C.
trachomatis GAGAgcTAAT GcaaGCcGTc GATGAtAAT...GAAc GATgTgGAAa
GaGGAatgGT TGT 22 -- G. vaginalis AGGAAcTcAT GaagGCTGTT
GACGAgTAC...TACc GACgTtGAgC GTGGtCAgGT TGT 135.sup.a -- S. aureus
TAGAATTART GGaaGCTGTa GATactTAC...TGAA GACgTaCAAC GTGGtCAAGT ATT
179 -- S. pneumoniae TGGAATTgAT GaacaCAGTT GATGAgTAT...TGAt
GAaATCGAAC GTGGACAAGT TAT 145.sup.a -- A. adiacens TAGAATTAAT
GGCTGCTGTT GACGAATAC...TGAA aACATCGAAC GTGGACAAGT TCT 118.sup.a --
G. haemolysans TCGAATTAAT GGaaaCAGTT GACGAATAC...TGAA GACATCGAAC
GTGGACAAGT TTT 87 -- G. morbillorum TCGAATTAAT GGaaaCAGTT
GACGAgTAC...TGAA GATATCGAAC GTGGACAAGT TTT 88 -- Selected sequence
for AATTAAT GGCTGCWGTT GAYGAA 1137 amplification primer Selected
sequence for A GAYATCSAAC GTGGACAAGT 1136 amplification
primer.sup.b The sequence numbering refers to the Enterococcus
durans tuf gene fragment (SEQ ID NO. 61). Nucleotides in capitals
are identical to the selected sequences or match those sequences.
Mismatches are indicated by lower-case letters. Dots indicate gaps
in the sequences displayed. "Y" "W" and "S" designate nucleotide
positions which are degenerated. "Y" stands for C or T; "W" stands
for A or T; "S" stands for C or G. "I" stands for inosine which is
a nucleotide analog that can bind to any of the four nucleotides A,
C, G or T. .sup.aThe SEQ ID NO. refers to previous patent
publication WO98/20157. .sup.bThis sequence is the
reverse-complement of the selected primer.
TABLE-US-00057 Annex XVIII: Strategy for the selection of the
Enterococcus faecalis-specific hybridization probe, of the
Enterococcus faecium-specific hybridization probe and of the
Enterococcus casseliflavus-flavescens-gallinarum group-specific
hybridization probe from tuf sequences. 395 448...526 549 SEQ ID
NO.: Accession #: E. avium GTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT
TGAAaTcGTa GGTATcGCT...CATc GGTGCtTTGt TACGTGGTGT 131.sup.a -- E.
casseliflavus GTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTT
GGTATTGCT...CATT GGTGCATTGC TACGTGGTGT 58 -- E. cecorum GTTGA
ACGTGGacAA GTaCGtGTTG GTGACGAAGT TGAAaTaGTT GGTATcCAT...CATc
GGTGCATTat TACGTGGTGT 59 -- E. dispar GTTGA ACGTGGacAA GTTCGCGTTG
GTGACGAAGT TGAAaTcGTa GGTATcGCT...CATT GGTGCATTat TACGTGGTGT 60 --
E. durans GTTGA ACGTGGacAA GTTCGCGTTG GTGACGttGT aGAtaTcGTT
GGTATcGCA...CATT GGTGCtTTaC TACGTGGTGT 61 -- E. faecalis GTTGA
ACGTGGTGAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTT GGTATTAAA...CTTc
GGTGCtTTat TACGTGGTGT 62 -- E. faecium GTTGA ACGTGGacAA GTTCGCGTTG
GTGACGAAGT TGAAGTTGTT GGTATTGCT...CATT GGTGCtTTaC TACGTGGTGT 608 --
E. flavescens GTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTT
GGTATTGCT...CATT GGTGCATTGC TACGTGGGGT 65 -- E. gallinarum GTTGA
ACGTGGacAA GTTCGCGTTG GTGATGAAGT aGAAaTcGTT GGTATTGCT...CATT
GGTGCATTGC TACGTGGGGT 609 -- E. hirae GTTGA ACGTGGacAA GTTCGCGTTG
GTGACGttGT aGAtaTcGTT GGTATcGCA...CATT GGTGCtTTaC TACGTGGTGT 67 --
E. mundtii GTTGA ACGTGGacAA GYTCGtGTTG GTGACGttaT cGAtaTcGTT
GGTATcGCA...CATT GGTGCgTTaC TACGTGGTGT 68 -- E. pseudoavium GTTGA
ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTa GGTATCGCT...CATc
GGTGCATTat TACGTGGTGT 69 -- E. raffinosus GTTGA ACGTGGacAA
GTTCGCGTTG GTGACGAAGT TGAAaTcGTa GGTATTGCT...CATT GGTGCATTat
TACGTGGTGT 70 -- E. saccharolyticus GTTGA ACGTGGacAA GTTCGCGTTG
GTGACGttGT aGAAaTcGTT GGTATcGAC...CATc GGTGCtTTat TACGTGGGGT 71 --
E. solitarius GTTGA ACGcGGgact aTcaaaGTCG GCGATGAAGT TGAcaTTaTT
GGTATTCAT...CATT GGTaCtTTGt TACGTGGTGT 72 -- C. diphtheriae GTTGA
gCGTGGctcc cTgaagGTCA ACGAGGAcGT cGAgaTcaTc GGTATcCGC...CTGT
GGTctgcTtC TcCGTGGCGT 662 -- G. vaginalis GTTGA gCGTGGTaAg
cTcCcaATCA ACACCCcAGT TGAgaTcGTT GGTtTgCGC...CACT GGTcttcTtC
TcCGcGGTAT 135.sup.a -- B. cepacia GTCGA gCGcGGcatc GTgaagGTCG
GCGAAGAAaT cGAAaTcGTc GGTATcAAG...CGTT GGTatccTGC TgCGcGGCAC 16 --
S. aureus GTTGA ACGTGGTcAA aTcaaaGTTG GTGAAGAAGT TGAAaTcaTc
GGTtTaCAT...CATT GGTGCATTat TACGTGGTGT 179 -- B. subtilis GTAGA
ACGcGGacAA GTTaaaGTCG GTGACGAAGT TGAAaTcaTc GGTcTTCAA...CATT
GGTGCccTtC TtCGcGGTGT -- Z99104 S. pneumoniae ATCGA cCGTGGTatc
GTTaaaGTCA ACGACGAAaT cGAAaTcGTT GGTATcAAA...CGTa GGTGtccTtC
TtCGTGGTGT 145.sup.a -- E. coli GTAGA ACGcGGTatc aTcaaaGTTG
GTGAAGAAGT TGAAaTcGTT GGTATcAAA...CGTa GGTGttcTGC TgCGTGGTAT 78 --
B. fragilis ATCGA AacTGGTGtt aTcCatGTAG GTGATGAAaT cGAAaTccTc
GGTtTgGGT...CGTa GGTctgTTGC TtCGTGGTGT -- M22247 C. trachomatis
ATTGA gCGTGGaatt GTTaaaGTTT CCGATAAAGT TcAgtTgGTc GGTcTTAGA...CGTT
GGattgcTcC TcaGaGGTAT 22 -- Selected sequences for GA ACGTGGTGAA
GTTCGC (E. faecalis) 1174 species-specific or AAGT TGAAGTTGTT
GGTATT (E. faecium) 602 group-specific T GGTGCATTGC TACGTGG 1122
hybridization probes The sequence numbering refers to the
Enterococcus faecium tuf gene fragments (SEQ ID NO. 608).
Nucleotides in capitals are identical to the selected sequences or
match those sequences. Mismatches are indicated by lower-case
letters. Dots indicate gaps in the sequences displayed. .sup.aThe
SEQ ID NO. refers to previous patent publication WO98/20157.
TABLE-US-00058 Annex XIX: Strategy for the selection of primers for
the identification of platelets contaminants from tuf sequences.
SEQ Accession 467 495 689 717 ID NO.: #: B. cereus GTA ACTGGTGTaG
AGATGTTCCG TAAACT...C AGTTCTACTT CCGTACAACT GACGTAAC 7 -- B.
subtilis GTT ACaGGTGTTG AAATGTTCCG TAAGCT...C AGTTCTACTT CCGTACAACT
GACGTAAC -- Z99104 E. cloacae TGT ACTGGCGTTG AAATGTTCCG CAAACT...C
AGTTCTACTT CCGTACAACT GACGTGAC 54 -- E. coli TGT ACTGGCGTTG
AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACTACT GACGTGAC 78 -- K.
oxytoca TGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACAACT
GACGTGAC 100 -- K. pneumoniae TGT ACTGGCGTTG AAATGTTCCG CAAACT...C
AGTTCTACTT CCGTACTACT GACGTGAC 103 -- P. aeruginosa TGC ACcGGCGTTG
AAATGTTCCG CAAGCT...C AGTTCTACTT CCGTACCACK GACGTGAC 153 -- S.
agalactiae GTT ACTGGTGTTG AAATGTTCCG TAAACA...C AATTCTACTT
CCGTACAACT GACGTAAC 209 -- S. aureus GTT ACaGGTGTTG AAATGTTCCG
TAAATT...C AATTCTATTT CCGTACTACT GACGTAAC 140.sup.a -- S.
choleraesuis TGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT
CCGTACTACT GACGTGAC 159 -- S. epidermidis GTT ACTGGTGTaG AAATGTTCCG
TAAATT...C AATTCTATTT CCGTACTACT GACGTAAC 611 -- S. marcescens TGT
ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACCACT GACGTGAC 168
-- S. mutans GTT ACTGGTGTTG AAATGTTCCG TAAACA...C AATTCTACTT
CCGTACAACT GACGTAAC 224 -- S. pyogenes GTT ACTGGTGTTG AAATGTTCCG
TAAACA...C AATTCTACTT CCGTACAACT GACGTAAC -- U40453 S. salivarius
GTT ACTGGTGTTG AAATGTTCCG TAAACA...C AGTTCTACTT CCGTACAACT GACGTAAC
146.sup.a -- S. sanguinis GTT ACTGGTGTTG AAATGTTCCG TAAACA...C
AGTTCTACTT CCGTACAACT GACGTTAC 227 -- Y. enterocolitica TGT
ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACAACT GAtGTAAC 235
-- Selected sequence ACTGGYGTTG AIATGTTCCG YAA 636 for
amplification primer Selected sequence TTCTAYTT CCGTACIACT GACGT
637 for amplification primer.sup.b The sequence numbering refers to
the E. coli tuf gene fragment (SEQ ID NO. 78). Nucleotides in
capitals are identical to the selected sequences or match those
sequences. Mismatches are indicated by lower-case letters. Dots
indicate gaps in the sequences displayed. "R" "Y" "M" "K" "W" and
"S" designate nucleotide positions which are degenerated. "R"
stands for A or G; "Y" stands for C or T; "M" stands for A or C;
"K" stands for G or T; "W" stands for A or T; "S" stands for C or
G. "I" stands for inosine which is a nucleotide analog that can
bind to any of the four nucleotides A, C, G or T. .sup.aThe SEQ ID
NO. refers to previous patent publication WO98/20157. .sup.bThis
sequence is the reverse-complement of the selected primer.
TABLE-US-00059 Annex XX: Strategy for the selection of the
universal amplification primers from atpD sequences. 616 657 781
812 SEQ ID NO. ACCESSION #: C. glutamicum GTGTTCGGTC AGATGGATGA
GCCACCAGGA GTCCGTATG CGC...CGTATg CCTTCCGCCG TGGGTTACCA GCCAAC --
X76875 M. tuberculosis GTATTCGGAC AGATGGACGA GCCGCCGGGC aCCCGTATG
CGT...CGGATg CCGTCGGCCG TGGGATACCA GCCCAC -- Z73419 E. faecalis
GTGTTCGGAC AAATGAACGA ACCACCAGGT GCTCGGATG CGG...CGTATg CCTTCTGCCG
TTGGTTACCA ACCAAC 291 -- S. agalactiae GTCTTTGGTC AAATGAATGA
ACCACCAGGA GCACGTATG CGT...CGTATg CCTTCAGCCG TTGGTTATCA ACCAAC 380
-- B. subtilis GTATTCGGAC AAATGAACGA GCCGCCGGGC GCACGTATG
CGT...CGTATg CCTTCAGCGG TTGGTTATCA GCCGAC -- Z28592 L.
monocytogenes GTATTCGGTC AAATGAACGA GCCACCAGGT GCGCGTATG
CGT...CGTATg CCATCTGCGG TAGGTTACCA ACCAAC 324 -- S. aureus
GTATTCGGGC AAATGAATGA GCCACCTGGT GCACGTATG CGT...CGTATg CCTTCTGCAG
TAGGTTACCA ACCAAC 366 -- A. baumannii GTCTACGGTC AGATGAACGA
GCCACCAGGT aaCCGTtTa CGC...CGTATg CCATCTGCGG TAGGTTACCA ACCTAC 243
-- N. gonorrhoeae GTGTATGGCC AAATGAACGA ACCTCCAGGC aaCCGTcTG
CGC...CGTATg CCTTCTGCAG TGGGTTACCA ACCGAC -- Genome project C.
freundii GTATATGGCC AGATGAACGA GCCGCCTGGA aaCCGTcTG CGT...CGTATg
CCATCAGCGG TAGGCTACCA GCCGAC 264 -- E. cloacae GTTTACGGCC
AGATGAACGA GCCACCAGGA aaCCGTcTG CGC...CGTATg CCTTCAGCGG TAGGTTATCA
GCCTAC 284 -- E. coli GTGTATGGCC AGATGAACGA GCCGCCGGGA aaCCGTcTG
CGC...CGTATg CCTTCAGCGG TAGGTTATCA GCCGAC 669 V00267 S. typhimurium
GTGTATGGCC AGATGAACGA GCCGCCGGGA aaCCGTcTG CGC...CGTATg CCTTCCGCAG
TAGGTTACCA GCCGAC 351 -- K. pneumoniae GTGTACGGCC AGATGAACGA
GCCGCCGGGA aaCCGTcTG CGC...CGTATg CCTTCAGCGG TAGGTTATCA GCCGAC 317
-- S. marcescens GTTTACGGCC AGATGAACGA GCCACCAGGT aaCCGTcTG
CGC...CGTATg CCATCCGCGG TAGGTTATCA GCCAAC 357 -- Y. enterocolitica
GTTTATGGCC AAATGAATGA GCCACCAGGT aaCCGTcTG CGC...CGTATg CCATCTGCCG
TAGGTTACCA GCCAAC 393 -- B. cepacia GTGTACGGCC AGATGAACGA
GCCGCCGGGC aaCCGTcTG CGC...CGTATg CCGTCGGCAG TGGGCTATCA GCCGAC --
X76877 H. influenzae GTTTATGGTC AAATGAACGA GCCACCAGGT aaCCGTtTa
CGT...CGTATg CCATCCGCGG TAGGTTACCA ACCGAC -- U32730 M. pneumoniae
GTGTTTGGTC AGATGAACGA ACCCCCAGGA GCACGGATG CGG...CGGATg CCATCAGCCG
TGGGTTACCA ACCAAC -- U43738 H. pylori TGCTATGGGC AAATGAATGA
GCCACCAGGT GCAAGGAat CGC...CGTATC CCTTCAGCGG TGGGGTATCA GCCCAC 670
V00267 B. fragilis GTGTTCGGAC AGATGAACGA ACCTCCTGGA GCACGTgct
TCA...CGTATg CCTTCTGCGG TAGGTTATCA ACCTAC -- M22247 Selected
sequences for C ARATGRAYGA RCCICCIGGI GYIMGIATG 562 universal
primers TAYGGIC ARATGAAYGARCCICCIGGI AA 564 Selected sequences for
ATH CCITCIGCIG TIGGITAYCA RCC 565 universal primers.sup.a ATG
CCITCIGCIG TIGGITAYCA RCC 563 The sequence numbering refers to the
Escherichia coli atpD gene fragment (SEQ ID NO. 669). Nucleotides
in capitals are identical to the selected sequences or match those
sequences. Mismatches for SEQ ID NOs. 562 and 565 are indicated by
lower-case letters. Mismatches for SEQ ID NOs. 564 and 563 are
indicated by underlined nucleotides. Dots indicate gaps in the
sequences displayed. "R" "Y" "M" "K" "W" and "S" letters designate
nucleotide positions which are degenerated. "R" stands for A or G;
"Y" stands for C or T; "M" stands for A or C; "K" stands for G or
T; "W" stands for A or T; "H" stands for A, C or T; "S" stands for
C or G. "I" stands for inosine which is a nucleotide analog that
can bind to any of the four nucleotides A, C, G or T. .sup.aThese
sequences are the reverse-complement of the selected primers.
TABLE-US-00060 Annex XXI: Specific and ubiquitous primers for
nucleic acid amplification (recA sequences). Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Universal primers (recA) 919 5'-GGI CCI GAR TCI TMI GGI
AAR AC 918.sup.a 437-459 920.sup.b 5'-TCI CCV ATI TCI CCI TCI AIY
TC 918.sup.a 701-723 921 5'-TIY RTI GAY GCI GAR CAI GC 918.sup.a
515-534 922.sup.b 5'-TAR AAY TTI ARI GCI YKI CCI CC 918.sup.a
872-894 Sequencing primers (recA) 1605 5'-ATY ATY GAA RTI TAY GCI
CC 1704.sup.a 220-239 1606 5'-CCR AAC ATI AYI CCI ACT TTT TC
1704.sup.a 628-650 Universal primers (rad51) 935 5'-GGI AAR WSI CAR
YTI TGY CAY AC 939.sup.a 568-590 936.sup.b 5'-TCI SIY TCI GGI ARR
CAI GG 939.sup.a 1126-1145 Universal primers (dmc1) 937 5'-ATI ACI
GAR GYI TTY GGI GAR TT 940.sup.a 1038-1060 938.sup.b 5'-CYI GTI GYI
SWI GCR TGI GC 940.sup.a 1554-1573 .sup.aSequences from databases.
.sup.bThese sequences are from the complementary DNA strand of the
sequence of the originating fragment given in the Sequence
Listing.
TABLE-US-00061 Annex XXII: Specific and ubiquitous primers for
nucleic acid amplification (speA sequences). Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Bacterial species: Streptococcus pyogenes 994 5'-TGG ACT
AAC AAT CTC GCA AGA GG 993.sup.a 60-82 995.sup.b 5'-ACA TTC TCG TGA
GTA ACA GGG T 993.sup.a 173-194 996 5'-ACA AAT CAT GAA GGG AAT CAT
TTA G 993.sup.a 400-424 997.sup.b 5'-CTA ATT CTT GAG CAG TTA CCA TT
993.sup.a 504-526 998 5'-GGA GGG GTA ACA AAT CAT GAA GG 993.sup.a
391-413 997.sup.b 5'-CTA ATT CTT GAG CAG TTA CCA TT 993.sup.a
504-526 .sup.aSequence from databases. .sup.bThese sequences are
from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
TABLE-US-00062 Annex XXIII: First strategy for the selection of
Streptococcus pyogenes-specific amplification primers from speA
sequences. ACCESSION # 57 85 170 197 SEQ ID NO.: speA X61573 CCTT
GGgCTAACAA cCTCaCAAGA aGTAT...GTGAtCCT.GT cgtTCAtGAG AATGTAAA --
speA AF029051 ~~~~ GGgCTAACAA cCTCaCAAGA aGTAT...GTGAtCCT.GT
cgtTCAtGAG AATGTAAA -- speA X61571 TCTT GGACTAACAA TCTCGCAAGA
GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA -- speA X61570 TCTT
GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA --
speA X61568 TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT
TACTCACGAG AATGTGAA -- speA X61569 TCTT GGACTAACAA TCTCGCAAGA
GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA -- speA X61572 TCTT
GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA --
speA X61560 TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT
TACTCACGAG AATGTGAA -- speA U40453 TCTT GGACTAACAA TCTCGCAAGA
GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 993 speA X61554 TCTT
GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA --
speA X61557 TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT
TACTCACGAG AATGTGAA -- speA X61559 TCTT GGACTAACAA TCTCGCAAGA
GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA -- speA X61558 TCTT
GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA --
speA X61556 TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT
TACTCACGAG AATGTGAA -- speA X61555 TCTT GGACTAACAA TCTCGCAAGA
GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA -- speA X61560 TCTT
GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA --
speA X61561 TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT
TACTCACGAG AATGTGAA -- speA X61566 TCTT GGACTAACAA TCTCGCAAGA
GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA -- speA X61567 TCTT
GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA --
speA X61562 TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT
TACTCACGAG AATGTGAA -- speA X61563 TCTT GGACTAACAA TCTCGCAAGA
GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA -- speA X61564 TCTT
GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA --
speA X61565 TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT
TACTCACGAG AATGTGAA -- speA AF055698 ~~~~ GGACTAACAA TCTCGCAAGA
GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA -- speA X03929.sup.a TCTT
GGACTAACAA TCTtGCcAaA aGGTA...GTGACCCTGGT TACTCACGAG AATGTGAA --
Selected T GGACTAACAA TCTCGCAAGA GG 994 sequence for
species-specific primer Selected ACCCT.GT TACTCACGAG AATGT 995
sequence for species-specific primer.sup.b The sequence numbering
refers to the Streptococcus pyogenes speA gene fragment (SEQ ID NO.
993). Nucleotides in capitals are identical to the selected
sequences or match those sequences. Mismatches are indicated by
lower-case letters. "~" indicate incomplete sequence data. Dots
indicate gaps in the sequences displayed. .sup.aThe extra G
nucleotide introducing a gap in the sequence is probably a
sequencing error. .sup.bThis sequence is the reverse-complement of
the selected primer.
TABLE-US-00063 Annex XXIV: Second strategy for the selection of
Streptococcus pyogenes-specific amplification primers from speA
sequences. SEQ ID Accession # 388 427 501 529 NO.: speA X61573 TA
TGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA
GAATTAGACT -- speA AF029051 TA TGGAGGGGTA ACAAATCATG AAGGGAATCA
TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT -- speA X61571 TA
CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA
GAATTAGACT -- speA X61570 TA CGGAGGGGTA ACAAATCATG AAGGGAATCA
TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT -- speA X61568 TA
CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA
GAATTAGACT -- speA X61569 TA CGGAGGGGTA ACAAATCATG AAGGGAATCA
TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT -- speA X61572 TA
CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA
GAATTAGACT -- speA X61560 TA CGGAGGGGTA ACAAATCATG AAGGGAATCA
TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT -- speA U40453 TA
CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA
GAATTAGACT 993 speA X61554 TA CGGAGGGGTA ACAAATCATG AAGGGAATCA
TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT -- speA X61557 TA
CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA
GAATTAGACT -- speA X61559 TA CGGAGGGGTA ACAAATCATG AAGGGAATCA
TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT -- speA X61558 TA
CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA
GAATTAGACT -- speA X61556 TA CGGAGGGGTA ACAAATCATG AAGGGAATCA
TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT -- speA X61555 TA
CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA
GAATTAGACT -- speA X61560 TA CGGAGGGGTA ACAAATCATG AAGGGAATCA
TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT -- speA X61561 TA
CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA
GAATTAGACT -- speA X61566 TA CGGAGGGGTA ACAAATCATG AAGGGAATCA
TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT -- speA X61567 TA
CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA
GAATTAGACT -- speA X61562 TA CGGAGGGGTA ACAAATCATG AAGGGAATCA
TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT -- speA X61563 TA
CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA
GAATTAGACT -- speA X61564 TA CGGAGGGGTA ACAAATCATG AAGGGAATCA
TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT -- speA X61565 TA
CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA
GAATTAGACT -- speA AF055698 TA CGGAGGGGTA ACAAATCATG AAGGGAATCA
TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT -- speA X03929 TA
CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA
GAATTAG.CT -- Selected GGAGGGGTA ACAAATCATG AAGG 998 sequences for
species-specific ACAAATCATG AAGGGAATCA TTTAG 996 primers Selected
AATGGT AACTGCTCAA GAATTAG 997 sequence for species-specific
primer.sup.a The sequence numbering refers to the Streptococcus
pyogenes speA gene fragment (SEQ ID NO. 993). Dots indicate gaps in
the sequences displayed. .sup.aThis sequence is the
reverse-complement of the selected primer.
TABLE-US-00064 Annex XXV: Strategy for the selection of
Streptococcus pyogenes-specific amplification primers from tuf
sequences. SEQ ID 140 186 619 647 NO.: S. anginosus A AGTTGACtTg
GTTGACGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATt cAtCCACACA
CTAAATT 211 S. bovis A AGTTGACCTT GTTGATGACG AAGAaTTGCT TGAATTgGTT
GAaATG...CC AgGTTCAATC cACCCACACA CTAAATT 212 S. dysgalactiae A
AATTGACCTT GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC
AACCCACABCA CTAAATT 217 S. pyogenes A AGTTGACCTT GTTGATGACG
AAGAGTTGCT TGAATTAGTT GAGATG...CC AAGTTCAATC AACCCACABCA CTAAATT
1002 S. agalactiae A AGTTGACCTT GTTGATGAtG AAGAaTTGCT TGAATTgGTT
GAaATG...CC AgGTTCAATC AACCCACABCA CTAAATT 144.sup.a S. oralis A
AATTGACtTg GTAGAcGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC
AACCCACABCA CTAAATT 985 S. pneumoniae A AGTTGACtTg GTTGAcGACG
AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACABCA CTAAATT
145.sup.a S. cristatus A GATCGACtTg GTTGATGACG AAGAaTTGCT
TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACABCA CTAAATT 215 S. mitis
A GATCGACtTg GTTGATGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC
AgGTTCAATC AACCCACABCA CTAAATT 982 S. gordonii A AGTTGACtTg
GTTGAcGAtG AAGAaTTGCT TGAGTTgGTT GAaATG...CC AgGTTCAATC AACCCACABCA
CTAAATT 200 S. sanguinis A AGTTGACtTg GTTGAcGAtG AAGAaTTGCT
TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACABCA CTAAATT 227 S.
parasanguinis A AGTTGACtTg GTTGATGAtG AAGAaTTGCT TGAATTgGTT
GAaATG...CC AgGTTCAATC AACCCACABCA CTAAATT 225 S. salivarius A
AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC TgGTTCAATC
AACCCACABCA CTAAATT 146.sup.a S. vestibularis A AGTTGACtTg
GTTGAcGAtG AAGAaTTGCTTGAATTgGTT GAaATG.. CC TgGTTCAATC AACCCACABCA
CTAAATT 231 S. suis A AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAgTTgGTT
GAaATG...CC AgGTTCtATC AACCCACABCA CTAAATT 229 S. mutans A
AGTTGAttTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATt
cACCCACACA CTAAATT 224 S. ratti A GGTTGACtTg GTTGATGAtG AAGAa TTGCT
TGAATTgGTT GAaATG...CC AgGTTCAATt cAtCCgCAcA CTAAATT 226 S. macacae
A AGTTGACtTa GTTGATGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC
AgGATCAATt cAtCCACAcA CTAAATT 222 S. cricetus A GGTTGACtTg
GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC TgGTTCAATC cAtCCACACA
CTAAATT 214 E. faecalis A AATgGAtaTg GTTGATGACG AAGAaTTatT
aGAATTAGTa GAaATG...CC AgcTaCAATC ActCCACACA CaAAATT 607 S. aureus
A AGTTGACaTg GTTGAcGAtG AAGAaTTatT aGAATTAGTa GAaATG...CC
TgGTTCAATt AcaCCACACA CTgAATT 176 B. cereus A ATgcGACaTg GTaGATGACG
AAGAaTTatT aGAATTAGTa GAaATG...AG CgGTTCtgTa AAagCtCACg CTAAATT 7
E. coli A ATgcGACaTg GTTGATGACG AAGAGcTGCT gGAAcTgGTT GAaATG...CC
GgGCaCcATC AAgCCgCACA CcAAGTT 78 Selected sequences for
species-specific TTGACCTT GTTGATGACG AAGAG 999 primers AAGAGTTGCT
TGAATTAGTT GAG 1001 Selected sequence for species-specific primer
AGTTCAATC AACCCACABCA CTAA 1000 .sup.b The sequence numbering
refers to the Streptococcus pyogenes tuf gene fragment (SEQ ID NO.
1002). Nucleotides in capitals are identical to the selected
sequences or match those sequences. Mismatches are indicated by
lower-case letters. Dots indicate gaps in the sequences displayed.
.sup.aThe SEQ ID NO. refers to previous patent publication
WO98/20157. .sup.bThis sequence is the reverse-complement of the
selected primer.
TABLE-US-00065 Annex XXVI: Strategy for the selection
stx.sub.1-specific amplification primers and hybridization probe.
SEQ ID Accession # 230 263 343 375 391 421 NO.: stx.sub.1 M19473a
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA -- stx.sub.1 M16625
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA -- stx.sub.1 M17358
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA -- stx.sub.1 Z36900
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
TTACCTTT...GTTACAT TGTCTaGTGA CAGTAGCTAT ACCA -- stx.sub.1 L04539
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA -- stx.sub.1 M19437
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA -- stx.sub.1 M24352
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA -- stx.sub.1 X07903
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA -- stx.sub.1 Z36899
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA -- stx.sub.1 Z36901
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA 1076 stx.sub.2 X61283
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 L11079
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 M21534
TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa
TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 M36727
TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa
TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 X81415
TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa
TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 X81416
TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa
TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 X81417
TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa
TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 X81418
TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa
TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 E03962
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 E03959
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 X07865
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 Y10775
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 Z37725
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA 1077 stx.sub.2 Z50754
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 X67514
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 L11078
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 X65949
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA -- stx.sub.2 AF043627
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACaG aTTTtCaGAT TTTgCACATa
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA -- Selected sequence
for ATGTC AGAGGGATAG ATCCAGAGGA AGG 1081 amplification primer
Selected sequence for CG CTTTGCTGAT TTTTCACATG TTACC 1084
hybridization probe Selected sequence for ACAT TGTCTGGTGA
CAGTAGCTAT A 1080 amplification primer.sup.a The sequence numbering
refers to the Escherichia coli stx.sub.1 gene fragment (SEQ ID NO.
1076). Nucleotides in capitals are identical to the selected
sequences or match those sequences. Mismatches are indicated by
lower-case letters. Dots indicate gaps in the sequences displayed.
.sup.aThis sequence is the reverse-complement of the selected
primer.
TABLE-US-00066 Annex XXVII: Strategy for the selection of
stx.sub.2-specific amplification primers and hybridization probe.
Accession # 543 570 614 641 684 708 SEQ ID NO.: stx.sub.1 M19473
AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC
gTtcTTA...A AGgtTgAGtA gCGTcCTgCC tGAC -- stx.sub.1 M16625 AGCga
TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A
AGgtTgAGtA gCGTcCTgCC tGAC -- stx.sub.1 M17358 AGCga TgtTaCGgTT
TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA
gCGTcCTgCC tGAC -- stx.sub.1 Z36900 AGCga TgtTaCGgTT TGTtACTGTG
ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC
tGAT -- stx.sub.1 L04539 AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC
ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAT --
stx.sub.1 M19437 AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt
ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAC -- stx.sub.1
M24352 AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC
gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAC -- stx.sub.1 X07903 AGCga
TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A
AGgtTgAGtA gTGTcCTgCC tGAC -- stx.sub.1 Z36899 AGCga TgtTaCGgTT
TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA
gTGTcCTgCC tGAC -- stx.sub.1 Z36901 AGCga TgtTaCGgTT TGTtACTGTG
ACA...CAAC ACTtgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC
tGAC 1076 stx.sub.2 X61283 AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC
ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG --
stx.sub.2 L11079 AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA.
..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG -- stx.sub.2
M21534 AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC
CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG -- stx.sub.2 M36727 AGCAG
TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G
AGAATCAGCA ATGTGCTTCC GGAG -- stx.sub.2 U72191 AGCAG TTCTGCGTTT
TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA
ATGTGCTTCC GGAG -- stx.sub.2 X81415 AGCAG TTCTGCGTTT TGTCACTGTC
ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC
GGAG -- stx.sub.2 X81416 AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC
ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG --
stx.sub.2 X81417 AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA.
..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG -- stx.sub.2
X81418 AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC
CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG -- stx.sub.2 E03962 AGCAG
TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G
CGAATCAGCA ATGTGCTTCC GGAG -- stx.sub.2 E03959 AGCAG TTCTGCGTTT
TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA
ATGTGCTTCC GGAG -- stx.sub.2 X07865 AGCAG TTCTGCGTTT TGTCACTGTC
ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC
GGAG -- stx.sub.2 Y10775 AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC
ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG --
stx.sub.2 Z37725 AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA.
..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG 1077 stx.sub.2
Z50754 AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC
CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG -- stx.sub.2 X67514 AGCAG
TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G
CGAATCAGCA ATGTGCTTCC GGAG -- stx.sub.2 L11078 AGCAG TTCTGCGTTT
TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G AGAATCAGCA
ATGTGCTTCC GGAG -- stx.sub.2 X65949 AGCAG TTCTGCGTTT TGTCACTGTC
ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G AGAATCAGCA ATGTGCTTCC
GGAG -- stx.sub.2 AF043627 AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC
ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG --
Selected sequence for amplification AG TTCTGCGTTT TGTCACTGTC 1078
primer Selected sequence for hybridization C ACTGTCTGA. ..AACTGCTC
CTGT 1085 probe Selected sequence for amplification AATCAGCA
ATGTGCTTCC G 1079 primer.sup.a The sequence numbering refers to the
Escherichia coli stx.sub.2 gene fragment (SEQ ID NO. 1077).
Nucleotides in capitals are identical to the selected sequences or
match those sequences. Mismatches are indicated by lower-case
letters. Dots indicate gaps in the sequences displayed. .sup.aThis
sequence is the reverse-complement of the selected primer.
TABLE-US-00067 Annex XXVIII: Strategy for the selection of
vanA-specific amplification primers from van sequences. Accession #
926 952 1230 1255 SEQ ID NO.: vanA X56895 GTCAAT AGCGCGGACG
AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1139 vanA M97297
GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1141
vanA -- GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA
TATG 1051 vanA -- GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG
CCCGTGTGGA TATG 1052 vanA -- GTCAAT AGCGCGGACG AATTGGACTA C...GT
AGAGGTCTAG CCCGTGTGGA TATG 1053 vanA -- GTCAAT AGCGCGGACG
AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1054 vanA -- GTCAAT
AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1055 vanA
-- GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG
1056 vanA -- GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG
CCCGTGTGGA TATG 1057 vanA -- GTCAAT AGCGCGGACG AATTGGACTA C...GT
AGAGGTCTAG CCCGTGTGGA TATG 1049 vanA -- GTCAAT AGCGCGGACG
AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1050 vanB U94526
GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT 1117
vanB U94527 GTAAAc AGtaCGGAaG AAcTaaACGC T...GC AGAGGgCTtG
CtCGTGTtGA TCTT -- vanB U94528 GTAAAc gGtaCGGAaG AAcTtaACGC T...GC
AGAGGgCTtG CCCGTGTtGA TCTT -- vanB U94529 GTAAAc gGtaCGGAaG
AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT -- vanB U94530 GTAAAc
gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT -- vanB
Z83305 GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA
TCTT -- vanB U81452 GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG
CCCGTGTtGA TCTT -- vanB U35369 GTAAAc AGtaCGGAaG AAcTaaACGC T...GC
AGAGGgCTtG CtCGTGTtGA TCTT -- vanB U72704 GTAAAc gGtaCGGAaG
AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT -- vanB L06138 GTAAAc
AGtaCGGAaG AAcTaaACGC T...GC AGAGGgCTtG CtCGTGTtGA TCTT -- vanB
L15304 GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA
TCTT -- vanB U00456 GTAAAc AGtaCGGAaG AAcTaaACGC T...GC AGAGGgCTtG
CtCGTGTtGA TCTT -- vanD AF130997 GTAtgc AagGCaGAaG AAcTGcAgGC
A...GC AGAGGatTgG CCCGcaTtGA cCTG -- vanE AF136925 GTAgAa
caaaaaagtG AtTTatAtAA A...GC AaAGGatTAG CgaGaaTcGA cTTT -- Selected
sequence for AAT AGCGCGGACG AATTGGAC 1090 amplification primer
Selected sequence for GAGGTCTAG CCCGTGTGGA T 1089 amplification
primer.sup.a The sequence numbering refers to the Enterococcus
faecium vanA gene fragment (SEQ ID NO. 1139). Nucleotides in
capitals are identical to the selected sequences or match those
sequences. Mismatches are indicated by lower-case letters. Dots
indicate gaps in the sequences displayed. .sup.aThis sequence is
the reverse-complement of the above selected primer.
TABLE-US-00068 Annex XXIX: Strategy for the selection of
vanB-specific amplification primers from van sequences. Accession #
470 495 608 633 SEQ ID NO.: vanA X56895 A CGCaATtGAA tCgGCAaGAC
AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1139 vanA M97297 A CGCaATtGAA
tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1141 vanA -- A
CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1051
vanA -- A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG
GAA 1052 vanA -- A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG
tATtCATCAG GAA 1053 vanA -- A CGCaATtGAA tCgGCAaGAC AATAT...ACG
GaATCTTtCG tATtCATCAG GAA 1054 vanA -- A CGCaATtGAA tCgGCAaGAC
AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1055 vanA -- A CGCaATtGAA
tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1056 vanA -- A
CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1057
vanA -- A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG
GAA 1049 vanA -- A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG
tATtCATCAG GAA 1050 vanB U94526 C TGCGATAGAA GCgGCAGGAC AATAT...ACG
GTATCTTCCG CATCCATCAG GAA 1117 vanB U94527 C TGCGATAGAA GCAGCAGGAC
AATAT...ACG GTATCTTCCG CATCCATCAG GAA -- vanB U94528 C TGCGATAGAA
GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA -- vanB U94529 C
TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA -- vanB
U94530 C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG
GAA -- vanB Z83305 C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG
CATCCATCAG GAA -- vanB U81452 C TGCGATAGAA GCgGCAGGAC AATAT...ACG
GTATCTTCCG CATCCATCAG GAA -- vanB U35369 C TGCGATAGAA GCAGCAGGAC
AATAT...ACG GTATCTTCCG CATCCATCAG GAA -- vanB U72704 C TGCGATAGAA
GCgGCAGGAC AATAT...ATG GTATCTTCCG CATCCATCAG GAA -- vanB L06138 C
TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA -- vanB
L15304 C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG
GAA -- vanB U00456 C TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG
CATCCATCAG GAA -- vanD AF130997 C AGCaATcGAA GaAGCAaGAa AATAT...ACG
GctTtTTtaa gATtCATCAG GAA -- vanE AF136925 A AGCaATAGAc GaAGCttcAa
AATAT...ATG GctTtTTCga CtatgAagAG AAA -- Selected sequence for
CGATAGAA GCAGCAGGAC AA 1095 amplification primer Selected sequence
for GTATCTTCCG CATCCATCAG 1096 amplification primer.sup.a The
sequence numbering refers to the Enterococcus faecium vanB gene
fragment (SEQ ID NO. 1117). Nucleotides in capitals are identical
to the selected sequences or match those sequences. Mismatches are
indicated by lower-case letters. Dots indicate gaps in the
sequences displayed. .sup.aThis sequence is the reverse-complement
of the above vanB sequence.
TABLE-US-00069 Annex XXX: Strategy for the selection of
vanC-specific amplification primers from vanC sequences. Accession
# 929 957 1064 1092 SEQ ID NO.: vanC1 -- GT CGACGGTTTT TTTGATTTTG
AAGAGAA...ACGGGTC TGGCTCGAAT CGATTTTTTC GT 1058 vanC1 -- GT
CGACGGTTTT TTTGATTTTG AAGAGAA...ACGGGTC TGGCTCGAAT CGATTTTTTC GT
1059 vanC1 M75132 GT CGACGGTTTT TTTGATTTTG AAGAGAA...ACGGGTC
TGGCTCGAAT CGATTTTTTC GT 1138 vanC2 -- GT AGACGGCTTT TTCGATTTTG
AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1060 vanC2 -- GT
AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT
1061 vanC2 -- GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT
CGACTTTTTT GT 1062 vanC2 -- GT AGACGGCTTT TTCGATTTTG
AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1063 vanC2 L29638 GT
AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT --
vanC2 L29638 GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT
CGACTTTTTT GT -- vanC3 -- GT AGACGGCTTT TTCGATTTTG
AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1064 vanC3 -- GT
AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT
1065 vanC3 -- GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGaC TTGCTCGCAT
CGACTTTTTT GT 1066 vanC3 L29639 GT AGACGGCTTT TTCGATTTTG
AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT -- Selected sequence
GACGGYTTT TTYGATTTTG AAGA 1101 for resistance primer Selected
sequence GGTC TKGCTCGMAT CGAYTTTTT 1102 for resistance primer.sup.a
The sequence numbering refers to the vanC1 gene fragment (SEQ ID
NO. 1138). Nucleotides in capitals are identical to the selected
sequences or match those sequences. Mismatches are indicated by
lower-case letters. Dots indicate gaps in the sequence displayed.
"R" "Y" "M" "K" "W" and "S" designate nucleotide positions which
are degenerated. "R" stands for A or G; "Y" stands for C or T; "M"
stands for A or C; "K" stands for G or T; "W" stands for A or T;
"S" stands for C or G. "I" stands for inosine which is a nucleotide
analog that can bind to any of the four nucleotides A, C, G or T.
.sup.aThis sequence is the reverse-complement of the selected
sequence.
TABLE-US-00070 Annex XXXI: Strategy for the selection of
Streptococcus pneumoniae-specific amplification primers and
hybridization probes from pbp1a sequences. SEQ ID Accession # 453
505 678 706 NO.: pbp1a M90528 A TTGACTAcCC AAGCATaCAc TATGCtAAtG
CtATTTCAAG TAATACAACC GA...TATATG ATGACaGAtA TGATGAAAAC CGT... --
pbp1a X67873 A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG
TAAcACAACC GA...TATATG ATGACCGAAA TGATGAAAAC AGT... -- pbp1a
AB006868 A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAAcACAACC
GA...TATATG ATGACCGACA TGATGAAAAC AGT... -- pbp1a AF046234 A
TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TATATG
ATGACCGAAA TGATGAAAAC TGT... -- pbp1a A TCGACTAcCC AAGtATtCAc
TActCAAAtG CCATTTCAAG TAAcACAACC GA...TATATG ATGACCGACA TGATGAAAAC
TGT... 1014 pbp1a A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG
TAAcACAACC GA...TACATG ATGACCGAAA TGATGAAAAC TGT... 1017 pbp1a
AB006873 A TCGACTAcCC AAGtcTtCAc TActCAAAtG CCATTTCAAG TAAcACAACC
GA...TATATG ATGACCGACA TGATGAAAAC AGT... -- pbp1a AF139883 A
TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG
ATGACCGACA TGATGAAAAC AGT... 1169 pbp1a A TCGACTATCC AAGCATGCAT
TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC
AGT... 1004 pbp1a A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG
TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT... 1007 pbp1a A
TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG
ATGACCGACA TGATGAAAAC AGT... 1008 pbp1a A TCGACTATCC AAGCATGCAT
TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC
AGT... 1009 pbp1a A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG
TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT... 1011 pbp1a
AF159448 A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA
GA...TATATG ATGACCGACA TGATGAAAAC AGT... -- pbp1a A TCGACTATCC
AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA
TGATGAAAAC TGT... 1005 pbp1a A TCGACTATCC AAGCATGCAT TATGCAAACG
CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA TGATGAAAAC TGT... 1015
pbp1a A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA
GA...TACATG ATGACCGAAA TGATGAAAAC TGT... 1006 pbp1a A TCGACTATCC
AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA
TGATGAAAAC TGT... 1012 pbp1a X67867 A TCGACTATCC AAGCATGCAT
TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA TGATGAAAAC
TGT... -- pbp1a A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG
TAAcACAACT GA...TATATG ATGACtGAAA TGATGAAAAC TGT... 1010 pbp1a
Z49094 A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT
GA...TATATG ATGACtGAAA TGATGAAAAC TGT... -- pbp1a A TCGACTATCC
AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG ATGACtGAAA
TGATGAAAAC TGT... 1013 pbp1a A TCGACTATCC AAGCATGCAT TATGCAAACG
CCATTTCAAG TAAcACAACT GA...TATATG ATGACtGAAA TGATGAAAAC TGT... 1016
pbp1a X67870 A TCGACTATCC AAGtATGCAT TAcGCAAACG CCATTTCAAG
TAAcACAACT GA...TATATG ATGACCGAAA TGATGAAAAC TGT... -- pbp1a A
TTGACTATCC AAGtATtCAc TActCAAAtG CtATTTCAAG TAATACAACT GA...TATATG
ATGACtGAAA TGATGAAAAC TGT... 1018 pbp1a AJ002290 A TTGAtTAcCC
AActATGgtc TATGCtAACG CtATTTCAAG TAATACAACT GA...TACATG ATGACtGAAA
TGATGAAAAC AGT... -- pbp1a X67871 A TCGACTAcCC AAGtcTtCAc
TActCAAAtG CCATTTCAAG TAAcACAACC GA...TACATG ATGACaGAAA TGATGAAAAC
AGT... -- Selected sequences for amplification primers GACTATCC
AAGCATGCAT TATG 1130 ATG ATGACHGAMA TGATGAAAAC 1129 Selected
sequence for hybridization probe CAAACG CCATTTCAAG TAATACAAC 1197
The sequence numbering refers to the Streptococcus pneumoniae pbp1a
gene fragment (SEQ ID NO. 1004). Nucleotides in capitals are
identical to the selected sequences or match those sequences.
Mismatches are indicated by lower-case letters. Dotes indicate gaps
in the sequences displayed. "R" "Y" "M" "K" "W" and "S" designate
nucleotide positions which are degenerated. "R" stands for A or G;
"Y" stands for C or T; "M" stands for A or C; "K" stands for G or
T; "W" stands for A or T; "H" stands for A, C or T; "S" stands for
C or G. "I" stands for inosine which is a nucleotide analog that
can bind to any of the four nucleotides A, C, G or T. SEQ ID
Accession # 756 783 813 840 SEQ ID NO.: pbp1a M90528 ...GCTGGTAA
aACtGGTACg TCTaACTATA...A ATACgGGTTA TGTAGCTCCG GAcGAAA -- pbp1a
X67873 ...GCTGGTAA aACAGGaACc TCTAACTATA...A CCtCTcaaTt TGTAGCaCCt
GATGAAC -- pbp1a AB006868 ...GCTGGTAA aACAGGaACc TCTAACTATA...A
CCtCTcaaTt TGTAGCaCCt GAcGAAC -- pbp1a AF046234 ...GCAGGTAA
aACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA -- pbp1a
...GCAGGTAA aACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA
1014 pbp1a ...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA
TGTAGCTCCA GATGAAA 1017 pbp1a AB006873 ...GCAGGTAA GACAGGTACT
TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA -- pbp1a AF139883
...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA
1169 pbp1a ...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA
TGTAGCTCCA GATGAAA 1004 pbp1a ...GCTGGTAA aACAGGaACc TCTAACTATA...A
ACACTGGCTA TGTAGCTCCA GATGAAA 1007 pbp1a ...GCTGGTAA aACAGGaACc
TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1008 pbp1a ...GCTGGTAA
aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1009 pbp1a
...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA
1011 pbp1a AF159448 ...GCTGGTAA aACAGGaACc TCTAACTATA...A
ACACTGGCTA TGTAGCTCCA GATGAAA -- pbp1a ...GCTGGTAA GACAGGTACT
TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1005 pbp1a ...GCTGGTAA
GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1015 pbp1a
...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA
1006 pbp1a ...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA
TGTAGCTCCA GATGAAA 1012 pbp1a X67867 ...GCTGGTAA GACAGGTACT
TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA -- pbp1a ...GCAGGTAA
GACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA 1010 pbp1a
Z49094 ...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA
GATGAAA -- pbp1a ...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA
CGTAGCTCCA GATGAAA 1013 pbp1a ...GCAGGTAA GACAGGTACT TCTAACTATA...A
ACACTGGCTA CGTAGCTCCA GATGAAA 1016 pbp1a X67870 ...GCAGGTAA
GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA -- pbp1a
...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA
1018 pbp1a AJ002290 ...GCAGGTAA GACgGGTACa TCTAACTACA...A
ACACTGGCTA C~~~~~~~~~ ~~~~~~~ -- pbp1a X67871 ...GCTGGTAA
aACAGGTACc TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA -- Selected
sequence for hybridization probe GGTAA GACAGGTACT TCTAACT 1193
Selected sequence for amplification primer.sup.a ACTGGYTA
YGTAGCTCCA GATG 1131 The sequence numbering refers to the
Streptococcus pneumoniae pbp1a gene fragment (SEQ ID NO. 1004).
Nucleotides in capitals are identical to the selected sequences or
match those sequences. Mismatches are indicated by lower-case
letters. Dots indicate gaps in the sequences displayed. "~"
indicates incomplete sequence data. "R" "Y" "W" and "S" designate
nucleotide positions which are degenerated. "R" stands for A or G;
"Y" stands for C or T; "W" stands for A or T; "S" stands for C or
G. "I" stands for inosine which is a nucleotide analog that can
bind to any of the four nucleotides A, C, G or T. .sup.aThis
sequence is the reverse-complement of the selected primer.
TABLE-US-00071 Annex XXXII: Specific and ubiquitous primers for
nucleic acid amplification (toxin sequences). Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Toxin gene: cdtA 2123 5'-TCT ACC ACT GAA GCA TTA C
2129.sup.a 442-460 2124.sup.b 5'-TAG GTA CTG TAG GTT TAT TG
2129.sup.a 580-599 Toxin gene: cdtB 2126 5'-ATA TCA GAG ACT GAT GAG
2130.sup.a 2665-2682 2127.sup.b 5'-TAG CAT ATT CAG AGA ATA TTG T
2130.sup.a 2746-2767 Toxin gene: stx.sub.1 1081 5'-ATG TCA GAG GGA
TAG ATC CA 1076.sup.a 233-252 1080.sup.b 5'-TAT AGC TAC TGT CAC CAG
ACA ATG T 1076.sup.a 394-418 Toxin gene: stx.sub.2 1078 5'-AGT TCT
GCG TTT TGT CAC TGT C 1077.sup.a 546-567 1079.sup.b 5'-CGG AAG CAC
ATT GCT GAT T 1077.sup.a 687-705 Toxin genes: stx.sub.1, and
stx.sub.2 1082 5'-TTG ARC RAA ATA ATT TAT ATG TG 1076.sup.a 278-300
1083.sup.b 5'-TGA TGA TGR CAA TTC AGT AT 1076.sup.a 781-800
.sup.aSequences from databases. .sup.bThese sequences are from the
complementary DNA strand of the sequence of the originating
fragment given in the Sequence Listing.
TABLE-US-00072 Annex XXXIII: Molecular beacon internal
hybridization probes for specific detection of toxin sequences.
Originating DNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide
sequence.sup.a NO. position Toxin gene: cdtA 2125.sup.b 5'-CAC GCG
GAT TTT GAA TCT CTT CCT CTA 2129.sup.c 462-488 GTA GCG CGT G Toxin
gene: cdtB 2128 5'-CAA CGC TGG AGA ATC TAT ATT TGT AGA 2130.sup.c
2714-2740 AAC TGC GTT G Toxin gene: stx.sub.1 1084 5'-CCA CGC CGC
TTT GCT GAT TTT TCA CAT 1076.sup.c 337-363 GTT ACC GCG TGG
2012.sup.d 5'-CCG CGG ATT ATT AAA CCG CCC TTC CGC 1076.sup.c
248-264 GG-MR-HEG-ATG TCA GAG GGA TAG ATC CA Toxin gene: stx.sub.2
1085 5'-CCA CGC CAC TGT CTG AAA CTG CTC CTG 1077.sup.c 617-638 TG
CGT GG .sup.aUnderlined nucleotides indicate the molecular beacon's
stem. .sup.bThese sequences are from the complementary DNA strand
of the sequence of the originating fragment given in the Sequence
Listing. .sup.cSequences from databases. .sup.dScorpion primer.
TABLE-US-00073 Annex XXXIV: Specific and ubiquitous primers for
nucleic acid amplification (van sequences). Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Resistance gene: vanA 1086 5'-CTA CTC CCG CCT TTT GGG TT
1049-1057.sup.a 513-532.sup.b 1087.sup.c 5'-CTC ACA GCC CGA AAC AGC
CT 1049-1057.sup.a 699-718.sup.b 1086 5'-CTA CTC CCG CCT TTT GGG TT
1049-1057.sup.a 513-532.sup.b 1088.sup.c 5'-TGC CGT TTC CTG TAT CCG
TC 1049-1057.sup.a 885-904.sup.b 1086 5'-CTA CTC CCG CCT TTT GGG TT
1049-1057.sup.a 513-532.sup.b 1089.sup.c 5'-ATC CAC ACG GGC TAG ACC
TC 1049-1057.sup.a 933-952.sup.b 1090 5'-AAT AGC GCG GAC GAA TTG
GAC 1049-1057.sup.a 629-649.sup.b 1091.sup.c 5'-AAC GCG GCA CTG TTT
CCC AA 1049-1057.sup.a 734-753.sup.b 1090 5'-AAT AGC GCG GAC GAA
TTG GAC 1049-1057.sup.a 629-649.sup.b 1089.sup.c 5'-ATC CAC ACG GGC
TAG ACC TC 1049-1057.sup.a 933-952.sup.b 1092 5'-TCG GCA AGA CAA
TAT GAC AGC 1049-1057.sup.a 662-682.sup.b 1088.sup.c 5'-TGC CGT TTC
CTG TAT CCG TC 1049-1057.sup.a 885-904.sup.b Resistance gene: vanB
1095 5'-CGA TAG AAG CAG CAG GAC AA 1117.sup.d 473-492 1096.sup.c
5'-CTG ATG GAT GCG GAA GAT AC 1117.sup.d 611-630 Resistance genes:
vanA, vanB 1112 5'-GGC TGY GAT ATT CAA AGC TC 1049-1057, 1117.sup.a
437-456.sup.b 1113.sup.c 5'-ACC GAC CTC ACA GCC CGA AA 1049-1057,
1117.sup.a 705-724.sup.b 1112 5'-GGC TGY GAT ATT CAA AGC TC
1049-1057, 1117.sup.a 437-456.sup.b 1114.sup.c 5'-TCW GAG CCT TTT
TCC GGC TCG 1049-1057, 1117.sup.a 817-837.sup.b 1115 5'-TTT CGG GCT
GTG AGG TCG GBT GHG CG 1049-1057, 1117.sup.a 705-730.sup.b
1114.sup.c 5'-TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117.sup.a
817-837.sup.b 1116 5'-TTT CGG GCT GTG AGG TCG GBT GHG CGG
1049-1057, 1117.sup.a 705-731.sup.b 1114.sup.c 5'-TCW GAG CCT TTT
TCC GGC TCG 1049-1057, 1117.sup.a 817-837.sup.b 1112 5'-GGC TGY GAT
ATT CAA AGC TC 1049-1057, 1117.sup.a 437-456.sup.b 1118.sup.c
5'-TTT TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117.sup.a
817-840.sup.b .sup.aThese sequences were aligned to derive the
corresponding primer. .sup.bThe nucleotide positions refer to the
vanA sequence fragment (SEQ ID NO. 1051). .sup.cThese sequences are
from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing. .sup.dSequences
from databases. Originating DNA fragment SEQ ID Nucleotide SEQ ID
NO. Nucleotide sequence NO. position 1115 5'-TTT CGG GCT GTG AGG
TCG GBT GHG CG 1049-1057, 1117.sup.a 705-730.sup.b 1118.sup.c
5'-TTT TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117.sup.a
817-840.sup.b 1116 5'-TTT CGG GCT GTG AGG TCG GBT GHG CGG
1049-1057, 1117.sup.a 705-731.sup.b 1118.sup.c 5'-TTT TCW GAG CCT
TTT TCC GGC TCG 1049-1057, 1117.sup.a 817-840.sup.b 1119 5'-TTT CGG
GCT GTG AGG TCG GBT GHG C 1049-1057, 1117.sup.a 705-729.sup.b
1118.sup.c 5'-TTT TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117.sup.a
817-840.sup.b 1120 5'-TTT CGG GCT GTG AGG TCG GBT GHG 1049-1057,
1117.sup.a 705-728.sup.b 1118.sup.c 5'-TTT TCW GAG CCT TTT TCC GGC
TCG 1049-1057, 1117.sup.a 817-840.sup.b 1121 5'-TGT TTG WAT TGT CYG
GYA TCC C 1049-1057, 1117.sup.a 408-429.sup.b 1111.sup.c 5'-CTT TTT
CCG GCT CGW YTT CCT GAT G 1049-1057, 1117.sup.a 806-830.sup.b 1112
5'-GGC TGY GAT ATT CAA AGC TC 1049-1057, 1117.sup.a 437-456.sup.b
1111.sup.c 5'-CTT TTT CCG GCT CGW YTT CCT GAT G 1049-1057,
1117.sup.a 806-830.sup.b 1123 5'-TTT CGG GCT GTG AGG TCG GBT G
1049-1057, 1117.sup.a 705-726.sup.b 1111.sup.c 5'-CTT TTT CCG GCT
CGW YTT CCT GAT G 1049-1057, 1117.sup.a 806-830.sup.b 1112 5'-GGC
TGY GAT ATT CAA AGC TC 1049-1057, 1117.sup.a 437-456.sup.b
1124.sup.c 5'-GAT TTG RTC CAC YTC GCC RAC A 1049-1057, 1117.sup.a
757-778.sup.b Resistance gene: vanC1 1103 5'-ATC CCG CTA TGA AAA
CGA TC 1058-1059.sup.a 519-538.sup.d 1104.sup.c 5'-GGA TCA ACA CAG
TAG AAC CG 1058-1059.sup.a 678-697.sup.d Resistance genes: vanC1,
vanC2, vanC3 1097 5'-TCY TCA AAA GGG ATC ACW AAA GTM AC
1058-1066.sup.a 607-632.sup.d 1098.sup.c 5'-TCT TCA AAA TCG AAA AAG
CCG TC 1058-1066.sup.a 787-809.sup.d 1099 5'-TCA AAA GGG ATC ACW
AAA GTM AC 1058-1066.sup.a 610-632.sup.d 1100.sup.c 5'-GTA AAK CCC
GGC ATR GTR TTG ATT TC 1058-1066.sup.a 976-1001.sup.d 1101 5'-GAC
GGY TTT TTY GAT TTT GAA GA 1058-1066.sup.a 787-809.sup.d 1102.sup.c
5'-AAA AAR TCG ATK CGA GCM AGA CC 1058-1066.sup.a 922-944.sup.d
Resistance genes: vanC2, vanC3 1105 5'-CTC CTA CGA TTC TCT TGA YAA
ATC A 1060-1066, 1140.sup.a 487-511.sup.e 1106.sup.c 5'-CAA CCG ATC
TCA ACA CCG GCA AT 1060-1066, 1140.sup.a 690-712.sup.e .sup.aThese
sequences were aligned to derive the corresponding primer.
.sup.bThe nucleotide positions refer to the vanA sequence fragment
(SEQ ID NO. 1051). .sup.cThese sequences are from the complementary
DNA strand of the sequence of the originating fragment given in the
Sequence Listing. .sup.dThe nucleotide positions refer to the vanC1
sequence fragment (SEQ ID NO. 1058). .sup.eThe nucleotide positions
refer to the vanC2 sequence fragment (SEQ ID NO. 1140). Originating
DNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Resistance gene: vanD 1591 5'-ATG AGG TAA TAG AAC GGA TT
1594 797-837 1592.sup.b 5'-CAG TAT TTC AGT AAG CGT AAA 1594 979-999
Resistance gene: vanE 1595 5'-AAA TAA TGC TCC ATC AAT TTG CTG A
1599.sup.a 74-98 1596.sup.b 5'-ATA GTC GAA AAA GCC ATC CAC AAG
1599.sup.a 394-417 1597 5'-GAT GAA TTT GCG AAA ATA CAT GGA
1599.sup.a 163-186 1598.sup.b 5'-CAG CCA ATT TCT ACC CCT TTC AC
1599.sup.a 319-341 Sequencing primers (vanAB) 1112 5'-GGC TGY GAT
ATT CAA AGC TC 1139.sup.a 737-756 1111.sup.b 5'-CTT TTT CCG GCT CGW
YTT CCT GAT G 1139.sup.a 1106-1130 Sequencing primers (vanA, vanX,
vanY) 1150 5'-TGA TAA TCA CAC CGC ATA CG 1141.sup.a 860-879
1151.sup.b 5'-TGC TGT CAT ATT GTC TTG CC 1141.sup.a 1549-1568 1152
5'-ATA AAG ATG ATA GGC CGG TG 1141.sup.a 1422-1441 1153.sup.b
5'-CTC GTA TGT CCC TAC AAT GC 1141.sup.a 2114-2133 1154 5'-GTT TGA
AGC ATA TAG CCT CG 1141.sup.a 2520-2539 1155.sup.b 5'-CAG TGC TTC
ATT AAC GTA GTC 1141.sup.a 3089-3109 Sequencing primers (vanC1)
1110 5'-ACG AGA AAG ACA ACA GGA AGA CC 1138.sup.a 122-144
1109.sup.b 5'-ACA TCG TGA TCG CTA AAA GGA GC 1138.sup.a 1315-1337
Sequencing primers (vanC2, vanC3) 1108 5'-GTA AGA ATC GGA AAA GCG
GAA GG 1140.sup.a 1-23 1107.sup.b 5'-CTC ATT TGA CTT CCT CCT TTG CT
1140.sup.a 1064-1086 .sup.aSequences from databases. .sup.bThese
sequences are from the complementary DNA strand of the sequence of
the originating fragment given in the Sequence Listing.
TABLE-US-00074 Annex XXXV: Internal hybridization probes for
specific detection of van sequences. Originating DNA fragment SEQ
ID Nucleotide SEQ ID NO. Nucleotide sequence NO. position
Resistance gene: vanA 1170 5'-ACG AAT TGG ACT ACG CAA TT
1049-1057.sup.a 639-658.sup.b 2292 5'-GAA TCG GCA AGA CAA TAT G
2293.sup.c 583-601 Resistance gene: vanB 1171 5'-ACG AGG ATG ATT
TGA TTG TC 1117.sup.c 560-579 2294 5'-AAA CGA GGA TGA TTT GAT TG
2296.sup.a 660-679 2295 5'-TTG AGC AAG CGA TTT CGG 2296.sup.a
614-631 Resistance gene: vanD 2297 5'-TTC AGG AGG GGG ATC GC
1594.sup.c 458-474 .sup.aThese sequences were aligned to derive the
corresponding primer. .sup.bThe nucleotide positions refer to the
vanA sequence fragment (SEQ ID NO. 1051). .sup.cSequences from
databases.
TABLE-US-00075 Annex XXXVI: Specific and ubiquitous primers for
nucleic acid amplification (pbp sequences). Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Resistance gene: pbp1a 1129 5'-ATG ATG ACH GAM ATG ATG AAA
AC 1004-1018.sup.a 681-703.sup.b 1131.sup.c 5'-CAT CTG GAG CTA CRT
ARC CAG T 1004-1018.sup.a 816-837.sup.b 1130 5'-GAC TAT CCA AGC ATG
CAT TAT G 1004-1018.sup.a 456-477.sup.b 1131 5'-CAT CTG GAG CTA CRT
ARC CAG T 1004-1018.sup.a 816-837.sup.b 2015 5'-CCA AGA AGC TCA AAA
ACA TCT G 2047.sup.d 909-930 2016.sup.c 5'-TAD CCT GTC CAW ACA GCC
AT 2047.sup.d 1777-1796 Sequencing primers (pbp1a) 1125 5'-ACT CAC
AAC TGG GAT GGA TG 1169.sup.d 873-892 1126.sup.c 5'-TTA TGG TTG TGC
TGG TTG AGG 1169.sup.d 2140-2160 1125 5'-ACT CAC AAC TGG GAT GGA TG
1169.sup.d 873-892 1128.sup.c 5'-GAC GAC YTT ATK GAT ATA CA
1169.sup.d 1499-1518 1127 5'-KCA AAY GCC ATT TCA AGT AA 1169.sup.d
1384-1403 1126.sup.c 5'-TTA TGG TTG TGC TGG TTG AGG 1169.sup.d
2140-2160 Sequencing primers (pbp2b) 1142 5'-GAT CCT CTA AAT GAT
TCT CAG GTG G 1172.sup.d 1-25 1143.sup.c 5'-CAA TTA GCT TAG CAA TAG
GTG TTG G 1172.sup.d 1481-1505 1142 5'-GAT CCT CTA AAT GAT TCT CAG
GTG G 1172.sup.d 1-25 1145.sup.c 5'-AAC ATA TTK GGT TGA TAG GT
1172.sup.d 793-812 1144 5'-TGT YTT CCA AGG TTC AGC TC 1172.sup.d
657-676 1143.sup.c 5'-CAA TTA GCT TAG CAA TAG GTG TTG G 1172.sup.d
1481-1505 Sequencing primers (pbp2x) 1146 5'-GGG ATT ACC TAT GCC
AAT ATG AT 1173.sup.d 219-241 1147.sup.c 5'-AGC TGT GTT AGC VCG AAC
ATC TTG 1173.sup.d 1938-1961 1146 5'-GGG ATT ACC TAT GCC AAT ATG AT
1173.sup.d 219-241 1149.sup.c 5'-TCC YAC WAT TTC TTT TTG WG
1173.sup.d 1231-1250 1148 5'-GAC TTT GTT TGG CGT GAT AT 1173.sup.d
711-730 1147.sup.c 5'-AGC TGT GTT AGC VCG AAC ATC TTG 1173.sup.d
1938-1961 .sup.aThese sequences were aligned to derive the
corresponding primer. .sup.bThe nucleotide positions refer to the
pbp1a sequence fragment (SEQ ID NO. 1004). .sup.cThese sequences
are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing. .sup.dSequences
from databases.
TABLE-US-00076 Annex XXXVII: Internal hybridization probes for
specific detection of pbp sequences. Originating DNA fragment SEQ
ID Nucleotide SEQ ID NO. Nucleotide sequence NO. position
Resistance gene: pbp1a 1132 5'-AGT GAA AAR ATG GCT GCT GC
1004-1018.sup.a 531-550.sup.b 1133 5'-CAT CAA GAA CAC TGG CTA YGT
AG 1004-1018.sup.a 806-828.sup.b 1134 5'-CTA GAT AGA GCT AAA ACC
TTC CT 1004-1018.sup.a 417-439.sup.b 1135 5'-CAT TAT GCA AAC GCC
ATT TCA AG 1004-1018.sup.a 471-493.sup.b 1192 5'-GGT AAA ACA GGA
ACC TCT AAC T 1004-1018.sup.a 759-780.sup.b 1193 5'-GGT AAG ACA GGT
ACT TCT AAC T 1004-1018.sup.a 759-780.sup.b 1194 5'-CAT TTC AAG TAA
TAC AAC AGA ATC 1004-1018.sup.a 485-508.sup.b 1195 5'-CAT TTC AAG
TAA CAC AAC TGA ATC 1004-1018.sup.a 485-508.sup.b 1196 5'-GCC ATT
TCA AGT AAT ACA ACA GAA 1004-1018.sup.a 483-506.sup.b 1197 5'-CAA
ACG CCA TTT CAA GTA ATA CAA C 1004-1018.sup.a 478-502.sup.b 1094
5'-GGT AAA ACA GGT ACT TCT AAC TA 1004-1018.sup.a 759-781.sup.b
1214 5'-GGT AAA ACA GGT ACC TCT AAC TA 1004-1018.sup.a
759-781.sup.b 1216 5'-GGT AAG ACT GGT ACA TCA AAC TA
1004-1018.sup.a 759-781.sup.b 1217 5'-CAA ATG CCA TTT CAA GTA ACA
CAA C 1004-1018.sup.a 478-502.sup.b 1218 5'-CAA ACG CCA TTT CAA GTA
ACA CAA C 1004-1018.sup.a 478-502.sup.b 1219 5'-CAA ATG CTA TTT CAA
GTA ATA CAA C 1004-1018.sup.a 478-502.sup.b 1220 5'-CAA ACG CCA TTT
CAA GTA ATA CGA C 1004-1018.sup.a 478-502.sup.b 2017 5'-ACT TTG AAT
AAG GTC GGT CTA G 2047.sup.c 1306-1327 2018 5'-ACA CTA AAC AAG GTT
GGT TTA G 2063 354-375 2019 5'-ACA CTA AAC AAG GTC GGT CTA G 2064
346-367 2020 5'-GTA GCT CCA GAT GAA ATG TTT G 2140.sup.c 1732-1753
2021 5'-GTA GCT CCA GAC GAA ATG TTT G 2057 831-852 2022 5'-GTA GCT
CCA GAT GAA ACG TTT G 2053.sup.c 805-826 2023 5'-GTA ACT CCA GAT
GAA ATG TTT G 2056 819-840 2024 5'-AGT GAA AAG ATG GCT GCT GC
2048.sup.c 1438-1457 2025 5'-AGT GAG AAA ATG GCT GCT GC 2047.sup.c
1438-1457 2026 5'-TCC AAG CAT GCA TTA TGC AAA CG 2047.sup.c
1368-1390 2027 5'-TCG GTC TAG ATA GAG CTA AAA CG 2047.sup.c
1319-1341 2028 5'-TAT GCT CTT CAA CAA TCA CG 2047.sup.c 1267-1286
2029 5'-AGC CGT TGA GAC TTT GAA TAA G 2047.sup.c 1296-1317 2030
5'-CTT AAT GGT CTT GGT ATC G 2047.sup.c 1345-1366 2031 5'-CGT GAC
TGG GGT TCT GCT ATG A 2049.sup.c 1096-1117 2032 5'-CGT GAC TGG GGA
TCA TCA ATG A 2047.sup.c 1096-1117 2033 5'-CGT GAC TGG GGT TCT GCC
ATG A 2057 195-216 2034 5'-ATC AAG AAC ACT GGC TAT GTA G 2050.sup.c
787-808 2035 5'-ATC AAG AAC ACT GGC TAC GTA G 2051.sup.c 787-808
2036 5'-ATC AAG AAC ACT GGT TAC GTA G 2047 1714-1735 2037 5'-ATC
AAA AAT ACT GGT TAT GTA G 2057 813-834 2038 5'-ATC AAG AAT ACT GGC
TAC GTA G 2052.sup.c 757-778 2039 5'-ATC AAA AAC ACT GGC TAT GTA G
2053.sup.c 787-808 .sup.aThese sequences were aligned to derive the
corresponding primer. .sup.bThe nucleotide positions refer to the
pbp1a sequence fragment (SEQ ID NO. 1004). .sup.cSequence from
databases.
TABLE-US-00077 Annex XXXVIII: Strategy for the selection of
vanAB-specific amplification primers and vanA- and vanB-specific
hybridization probes from van sequences. SEQ ID Accession # 734 759
936 961 NO.: vanA X56895 GTAGGCT GCGATATTCA AAGCTCAGC...CGGAACGAATT
GGACTACGCA ATTGAA... 1139 vanA M97297 GTAGGCT GCGATATTCA
AAGCTCAGC...CGGAACGAATT GGACTACGCA ATTGAA... 1141 vanA GTAGGCT
GCGATATTCA AAGCTCAGC...CGGAACGAATT GGACTACGCA ATTGAA... 1051 vanA
GTAGGCT GCGATATTCA AAGCTCAGC...CGGAACGAATT GGACTACGCA ATTGAA...
1052 vanA GTAGGCT GCGATATTCA AAGCTCAGC...CGGAACGAATT GGACTACGCA
ATTGAA... 1053 vanA GTAGGCT GCGATATTCA AAGCTCAGC...CGGAACGAATT
GGACTACGCA ATTGAA... 1054 vanA GTAGGCT GCGATATTCA
AAGCTCAGC...CGGAACGAATT GGACTACGCA ATTGAA... 1055 vanA GTAGGCT
GCGATATTCA AAGCTCAGC...CGGAACGAATT GGACTACGCA ATTGAA... 1056 vanA
GTAGGCT GCGATATTCA AAGCTCAGC...CGGAACGAATT GGACTACGCA ATTGAA...
1057 vanA GTAGGCT GCGATATTCA AAGCTCAGC...CGGAACGAATT GGACTACGCA
ATTGAA... 1049 vanA GTAGGCT GCGATATTCA AAGCTCAGC...CGGAACGAATT
GGACTACGCA ATTGAA... 1050 vanB U94526 GTGGGCT GTGATATTCA
AAGCTCCGC...CGGAaGAAcT taACgctGCg ATaGAA... 1117 vanB U94527
GTAGGCT GCGATATTCA AAGCTCCGC...CGGAaGAAcT aaACgctGCg ATaGAA... --
vanB U94528 GTGGGCT GTGATATTCA AAGCTCCGC...CGGAaGAAcT taACgctGCg
ATaGAA... -- vanB U94529 GTGGGCT GTGATATTCA AAGCTCCGC...CGGAaGAAcT
taACgctGCg ATaGAA... -- vanB U94530 GTGGGCT GTGATATTCA
AAGCTCCGC...CGGAaGAAcT taACgctGCg ATaGAA... -- vanB Z83305 GTGGGCT
GTGATATTCA AAGCTCCGC...CGGAaGAAcT taACgctGCg ATaGAA... -- vanB
U81452 GTGGGCT GTGATATTCA AAGCTCCGC...CGGAaGAAcT taACgctGCg
ATaGAA... -- vanB U35369 GTAGGCT GCGATATTCA AAGCTCCGC...CGGAaGAAcT
aaACgctGCg ATaGAA... -- vanB U72704 GTGGGCT GCGATATTCA
AAGCTCCGC...CGGAaGAAcT taACgctGCg ATaGAA... -- vanB L06138 GTAGGCT
GCGATATTCA AAGCTCCGC...CGGAaGAAcT aaACgctGCg ATaGAA... -- vanB
L15304 GTGGGCT GTGATATTCA AAGCTCCGC...CGGAaGAAcT taACgctGCg
ATaGAA... -- vanB U00456 GTAGGCT GCGATATTCA AAGCTCCGC...CGGAaGAAcT
aaACgctGCg ATaGAA... -- vanD AF130997 GTGGGaT GCGATATTCA
AAGCTCCGT...CAGAaGAAcT GcAggcaGCA ATcGAA... -- vanE AF136925
GTAGGtT GTGgTATcgg AgctgCAGC...AAAgtGAtTT atAtaAaGCA ATaGAC... --
Selected sequence GGCT GYGATATTCA AAGCTC 1112 for amplification
primer Selected sequence ACGAATT GGACTACGCA ATT (vanA) 1170 for
hybridization probe The sequence numbering refers to the
Enterococcus faecium vanA gene fragment (SEQ ID NO. 1139).
Nucleotides in capitals are identical to the selected sequences or
match those sequences. Mismatches are indicated by lower-case
letters. Dots indicate gaps in the sequences displayed. "R" "Y" "M"
"K" "W" and "S" designate nucleotide positions which are
degenerated. "R" stands for A or G; "Y" stands for C or T; "M"
stands for A or C; "K" stands for G or T; "W" stands for A or T;
"S" stands for C or G. "I" stands for inosine which is a nucleotide
analog that can bind to any of the four nucleotides A, C, G or T.
SEQ ID Accession # 1038 1063 1103 1133 NO.: vanA X56895 GAAACagt
GccGcGTTag TTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1139 vanA
M97297 GAAACagt GccGcgTTag TTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA
AAAAGGCT 1141 vanA GAAACagt GccGcgTTag TTGTtGGC...ATT CATCAGGAAG
TCGAGCCGGA AAAAGGCT 1051 vanA GAAACagt GccGcgTTag cTGTtGGC...ATT
CATCAGGAAG TCGAGCCGGA AAAAGGCT 1052 vanA GAAACagt GccGcgTTag
cTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1053 vanA GAAACagt
GccGcgTTag TTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1054 vanA
GAAACagt GccGcgTTag cTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT
1055 vanA GAAACagt GccGcgTTag cTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA
AAAAGGCT 1056 vanA GAAACagt GccGcgTTag cTGTtGGC...ATT CATCAGGAAG
TCGAGCCGGA AAAAGGCT 1057 vanA GAAACagt GccGcgTTag cTGTtGGC...ATT
CATCAGGAAG TCGAGCCGGA AAAAGGCT 1049 vanA GAAACagt GccGcgTTag
cTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1050 vanB U94526
GGAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
1117 vanB U94527 GAAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA
ACGAGCCGGA AAAAGGCT -- vanB U94528 GGAACGAG GATGATTTGA
TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT -- vanB U94529
GGAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
-- vanB U94530 GGAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA
ACGAGCCGGA AAAAGGCT -- vanB Z83305 GGAACGAG GATGATTTGA
TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT -- vanB U81452
GGAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
-- vanB U35369 GAAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA
ACGAGCCGGA AAAAGGCT -- vanB U72704 GGAACGAG GATGATTTGA
TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGAT -- vanB L06138
GAAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
-- vanB L15304 GGAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA
ACGAGCCGGA AAAAGGCT -- vanB U00456 GAAACGAG GATGATTTGA
TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT -- vanD AF130997
GAAACGga aATGATcTcA TgGctGGC...ATT CATCAGGAAG cacAGCCGGA aAAGGGAT
-- vanE AF136925 GGAA...t GAacAaTTGg TcGTtGGA...TAT gAagAGaAAt
ACaA...... ......TT -- Selected sequence ACGAG GATGATTTGA TTGTC
(vanB) 1171 for hybridization probe Selected sequence CATCAGGAAR
WCGAGCCGGA AAAAG 1111 for amplification prime.sup.a The sequence
numbering refers to the Enterococcus faecium vanA gene fragment
(SEQ ID NO. 1139). Nucleotides in capitals are identical to the
selected sequences or match those sequences. Mismatches are
indicated by lower-case letters. Dots indicate gaps in the
sequences displayed. "R" and "W" designate nucleotide positions
which are degenerated. "R" stands for A or G; "W" stands for A or T
.sup.aThis sequence is the reverse-complement of the above selected
primer.
TABLE-US-00078 Annex XXXIX: Internal hybridization probe for
specific detection of mecA. Originating DNA fragment SEQ ID SEQ ID
Nucleotide NO. Nucleotide sequence NO. position Resistance gene:
mecA 1177 5'-GCT CAA CAA GTT CCA 1178.sup.a 1313-1332 GAT TA
.sup.aSequence from databases.
TABLE-US-00079 Annex XL: Specific and ubiquitous primers for
nucleic acid amplification (hexA sequences). Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.
position Bacterial species: Streptococcus pneumoniae 1179 5'-ATT
TGG TGA CGG GTG ACT TT 1183.sup.a 431-450 1181.sup.b 5'-AGC AGC TTA
CTA GAT GCC GT 1183-1191.sup.c 652-671.sup.d Sequencing primers
1179 5'-ATT TGG TGA CGG GTG ACT TT 1183.sup.a 431-450 1182.sup.b
5'-AAC TGC AAG AGA TCC TTT GG 1183.sup.a 1045-1064 .sup.aSequences
from databases. .sup.bThese sequences are from the complementary
DNA strand of the sequence of the originating fragment given in the
Sequence Listing. .sup.cThese sequences were aligned to derive the
corresponding primer. .sup.dThe nucleotide positions refer to the
hexA sequence fragment (SEQ ID NO. 1183).
TABLE-US-00080 Annex XLI: Internal hybridization probe for specific
detection of hexA sequences. Originating DNA fragment SEQ SEQ ID
Nucleotide ID NO. Nucleotide sequence NO. position Bacterial
species: Streptococcus pneumoniae 1180.sup.a 5'-TCC ACC GTT GCC
1183-1191.sup.b 629-647.sup.c AAT CGC A .sup.aThis sequences is
from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing. .sup.bThese
sequences were aligned to derive the corresponding primer.
.sup.cThe nucleotide positions refer to the hexA sequence fragment
(SEQ ID NO. 1183).
TABLE-US-00081 Annex XLII: Strategy for the selection of
Streptococcus pneumoniae species-specific amplification primers and
hybridization probe from hexA sequences. 428 453 626 S. pneumoniae
TGG ATTTGGTGAC GGGTGACTTT TAT...ATTTG CGATTGGCAA CGGTGGAGCA S.
pneumoniae ~~~ ~~~~~~TGAC GGGTGACTTT TAT...ATTTG CGATTGGCAA
CGGTGGAGCA S. pneumoniae ~~~ ~~~~~~TGAC GGGTGACTTT TAT...ATTTG
CGATTGGCAA CGGTGGAGCA S. pneumoniae ~~~ ~~~~~~TGAC GGGTGACTTT
TAT...ATTTG CGATTGGCAA CGGTGGAGCA S. pneumoniae ~~~ ~~~~~~TGAC
GGGTGACTTT TAT...ATTTG CGATTGGCAA CGGTGGAGCA S. oralis ~~~
~~~~~~~~~~GGGTGACTTT TAT...ATCca CGAcTGGCAg CtGTGGAGCA S. mitis ~~~
~~~~GGTGAC GGGTGACTTT TAT...ATTca CGATTGGCAg CtGTGGAGCA S. mitis
~~~ ~~~~~~TGAC GGGTGACTTT CAG...GCGaG gagcTGtCtc CtaTGGAGCG S.
mitis ~~~ ~~~~~~TGAC GGGTGACTTT CAG...GCGaG gaAcTGtCtc CtaTGGAGCG
Selected sequence for ATTTGGTGAC GGGTGACTTT amplification primer
Selected sequences for amplification primers.sup.a Selected
sequence for TG CGATTGGCAA CGGTGGA hybridization probe.sup.a SEQ ID
674 1042 1067 NO.: S. pneumoniae AACGGCATCT AGTAAGCTGC
TCCA...AATCCAAAG GATCTCTTGC AGTTGGC 1183 S. pneumoniae AACGGCATCT
AGTAAGCTGC TCCA...AATCCAAAG GATCTCTTG~ ~~~~~~~ 1184 S. pneumoniae
AACGGCATCT AGTAAGCTGC TCCA...AATCCAAAG GATCTCT~~~ ~~~~~~~ 1185 S.
pneumoniae AACGGCATCT AGTAAGCTGC TCCA...AATCCAAAG GATCTCTT~~
~~~~~~~ 1186 S. pneumoniae AACGGCATCT AGTAAGCTGC TCCG...AATCCAAAG
GATCTCTT~~ ~~~~~~~ 1187 S. oralis AgCGGCAgCT AGTAAGCTcC
TCCA...~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~ 1188 S. mitis AgCGGCATCT
AGTAAaCTGC TTCA...AATCCAAAG GATCTCTT~~ ~~~~~~~ 1189 S. mitis
TcaGGCAgCa gGgAAaCTGC TGGA...~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~ 1190 S.
mitis TcaGGCAgCg gGgAAatTGC TAGA...AATCCAAAG GATCTCTT~~ ~~~~~~~
1191 Selected sequence for 1179 amplification primer Selected
sequences ACGGCATCT AGTAAGCTGC T 1181 for amplification CCAAAG
GATCTCTTGC AGTT 1182 primers.sup.a Selected sequence for 1180
hybridization probe.sup.a The sequence numbering refers to the
Streptococcus pneumoniae hexA gene fragment (SEQ ID NO. 1183).
Nucleotides in capitals are identical to the selected sequences or
match those sequences. Mismatches are indicated by lower-case
letters. Dots indicate gaps in the sequences displayed. "--"
indicate incomplete sequence data. .sup.aThis sequence is the
reverse-complement of the selected primer.
TABLE-US-00082 Annex XLIII: Specific and ubiquitous primers for
nucleic acid amplification (pcp sequence). Originating DNA fragment
SEQ SEQ ID Nucleotide ID NO. Nucleotide sequence NO. position
Bacterial species: Streptococcus pyogenes 1211 5'-ATT CTT GTA ACA
GGC TTT 1215.sup.a 291-314 GAT CCC 1210.sup.b 5'-ACC AGC TTG CCC
AAT ACA 1215.sup.a 473-494 AAG G .sup.aSequences from databases.
.sup.bThese sequences are from the complementary DNA strand of the
sequence of the originating fragment given in the Sequence
Listing.
TABLE-US-00083 Annex XLIV: Specific and ubiquitous primers for
nucleic acid amplification of S. saprophyticus sequences of unknown
coding potential. Originating DNA fragment SEQ ID SEQ ID Nucleotide
NO. Nucleotide sequence NO. position Bacterial species:
Staphylococcus saprophyticus 1208 5'-TCA AAA AGT TTT CTA 74, 1093,
169-193.sup.c AAA AAT TTA C 1198.sup.b 1209.sup.a 5'-ACG GGC GTC
CAC AAA 74, 1093, 355-379.sup.c ATC AAT AGG A 1198.sup.b .sup.aThis
sequence is from the complementary DNA strand of the sequence of
the originating fragment given in the Sequence Listing. .sup.bThese
sequences were aligned to derive the corresponding primer.
.sup.cThe nucleotide positions refer to the S. saprophyticus
unknown gene sequence fragment (SEQ ID NO. 1198).
TABLE-US-00084 Annex XLV: Molecular beacon internal hybridization
probes for specific detection of antimicrobial agents resistance
gene sequences. Originating DNA fragment SEQ ID Nucleotide SEQ ID
NO. Nucleotide sequence.sup.a NO. position Resistance gene: gyrA
2250 5'-CCG TCG GAT GGT GTC GTA TAC CGC GGA GTC 1954.sup.b 218-243
GCC GAC GG 2251 5'-CGG AGC CGT TCT CGC TGC GTT ACA TGC TGG
1954.sup.b 259-286 TGG CTC CG Resistance gene: mecA 1231 5'-GCG AGC
CCG AAG ATA AAA AAG AAC CTC TGC 1178.sup.b 1291-1315 TGC TCG C
Resistance gene: parC 1938.sup.b 5'-CCG CGC ACC ATT GCT TCG TAC ACT
GAG GAG 1321.sup.c 232-260 TCT CCG CGC GG 1939 5'-CGA CCC GGA TGG
TAG TAT CGA TAA TGA TCC 1321.sup.c 317-346 GCC AGC GGC CGG GTC G
1955.sup.b 5'-CGC GCA ACC ATT GCT TCG TAC ACT GAG GAG 1321.sup.c
235-260 TCT GCG CG Resistance gene: vanA 1239 5'-GCG AGC GCA GAC
CTT TCA GCA GAG GAG GCT 1051 860-880 CGC 1240 5'-GCG AGC CGG CAA
GAC AAT ATG ACA GCA AAA 1051 663-688 TCG CTC GC Resistance gene:
vanB 1241 5'-GCG AGC GGG GAA CGA GGA TGA TTT GAT TGG 1117 555-577
CTC GC Resistance gene: vanD 1593 5'-CCG AGC GATTTA CCG GAT ACT TGG
CTG ICG 1594 835-845 CTC GG .sup.aUnderlined nucleotides indicate
the molecular beacon's stem. .sup.bThis sequence is from the
complementary DNA strand of the sequence of the originating
fragment given in the Sequence Listing. .sup.cSequence from
databases.
TABLE-US-00085 Annex XLVI: Molecular beacon internal hybridization
probe for specific detection of S. aureus gene sequences of unknown
coding potential. Originating DNA fragment SEQ SEQ ID ID Nucleotide
NO. Nucleotide sequence.sup.a NO. position Bacterial species: S.
aureus 1232 5'-GGA GCC GCG CGA TTT TAT 1244 53-80 AAA TGA ATG TTG
ATA ACC GGC TCC .sup.aUnderlined nucleotides indicate the molecular
beacon's stem.
TABLE-US-00086 Annex XLVII: Molecular beacon internal hybridization
probes for specific detection of tuf sequences. Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence.sup.a NO.
position Bacterial species: Chlamydia pneumoniae 2091 5'-CGC GAC
TTG AGA TGG AAC TTA GTG AGC 20 157-183 TTC TTG GTC GCG 2092 5'-CGC
GAC GAA AGA ACT TCC TGA AGG TCG 20 491-516 TGC AGG TCC AG Bacterial
species: Chlamydia trachomatis 2213 5'-CGT GCC ATT GAC ATG ATT TCC
GAA GAA 1739.sup.b 412-441 GAC GCT GAA GGC ACG Bacterial species:
Enterococcus faecalis 1236 5'-GCG AGC CGT GGT GAA GTT CGC GTT GGT
883 370-391 GGC TCG C Bacterial species: Enterococcus faecium 1235
5'-GCG AGC CGA AGT TGA AGT TGT TGG TAT 64 412-437 TGC TGG CTC GC
Bacterial species: Legionella pneumophila 2084.sup.c 5'-CAC GCG TCA
ACA CCC GTA CAA GTC GTC 112 461-486 TTT TGC GCG TG Bacterial
species: Mycoplasma pneumoniae 2096.sup.c 5'-CGC GAC CGG TAC CAC
GGC CAG TAA TCG 2097.sup.b 658-679 TGT CGC G Bacterial species:
Neisseria gonorrhoeae 2177 5'-GGC ACG GAC AAA CCA TTC CTG CTG CCT
126 323-357 ATC GAA ACG TGT TCC CGT GCC 2178 5'-GGC ACG ACA AAC CAT
TCC TGC TGC CTA 126 323-348 TCG AAC GTG CC 2179 5'-GGC AGC TCT ACT
TCC GTA CCA CTG ACG 126 692-718 TAA CCG GCT GCC .sup.aUnderlined
nucleotides indicate the molecular beacon's stem. .sup.bSequence
from databases. .sup.cThis sequence is from the complementary DNA
strand of the sequence of the originating fragment given in the
Sequence Listing. Originating DNA fragment SEQ ID Nucleotide SEQ ID
NO. Nucleotide sequence.sup.a NO. position Bacterial species:
Pseudomonas aeruginosa 2122 5'-CCG AGC GAA TGT AGG AGT CCA GGG TCT
153, 280-302.sup.d CTG CTC GG 880, 2138.sup.b,c Bacterial species:
Staphylococcus aureus 2186 5'-ACG CGC TCA AAG CAG AAG TAT ACG TAT
1728 615-646 TAT CAA AAG ACG CGC GT Bacterial group: Staphylococcus
sp. other than S. aureus 1233 5'-GCG AGC GTT ACT GGT GTA GAA ATG
TTC 878 372-394 CGG CTC GC Fungal species: Candida albicans 2073
5'-CCG AGC AAC ATG ATT GAA CCA TCC ACC 408 404-429 AAC TGG CTC GG
Fungal species: Candida dubliniensis 2074 5'-CCG AGC AAC ATG ATT
GAA GCT TCC ACC 414 416-441 AAC TGG CTC GG Fungal species: Candida
glabrata 2110.sup.b 5'-GCG GGC CCT TAA CGA TTT CAG CGA ATC 417
307-335 TGG ATT CAG CCC GC 2111 5'-GCG GGC ATG TTG AAG CCA CCA CCA
ACG 417 419-447 CTT CCT GGC CCG C Fungal species: Candida krusei
2112.sup.b 5'-GCG GGC TTG ATG AAG TTT GGG TTT CCT 422 318-347 TGA
CAA TTG CCC GC 2113 5'-GCG GGC ACA AGG GTT GGA CTA AGG AAA 422
419-447 CCA AGG CAG CCC GC 2114 5'-GCG GGC ATC GAT GCT ATT GAA CCA
CCT 422 505-533 GTC AGA CCG CCC GC .sup.aUnderlined nucleotides
indicate the molecular beacon's stem. .sup.bSequence from
databases. .sup.cThese sequences were aligned to derive the
corresponding primer. .sup.dThe nucleotide positions refer to the
P. aeruginosa tuf sequence fragment (SEQ ID NO. 153). Originating
DNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence.sup.a
NO. position Fungal species: Candida lusitaniae 2115.sup.b 5'-GCG
GGC GGT AAG TCC ACC GGT AAG ACC 424 304-330 TTG TTG GCC CGC 2116
5'-GCG GGC GTA AGT CAC CGG TAA GAC CTT 424 476-502 GTT GGC CCG C
2117 5'-GCG GGC GAC GCC ATT GAG CCA CCT TCG 424 512-535 AGA GCC CGC
Fungal species: Candida parapsilosis 2118.sup.b 5'-GCG GGC TCC TTG
ACA ATT TCT TCG TAT 426 301-330 CTG TTC TTG GCC CGC Fungal species:
Candida tropicalis 2119 5'-GCG GGC TTA CAA CCC TAA GGC TGT TCC 429
357-384 ATT CGT TGC CCG C 2120 5'-GCG GGC AGA AAC CAA GGC TGG TAA
GGT 429 459-487 TAC CGG AGC CCG C Fungal species: Cryptococcus
neoformans 2106 5'-GCG AGC AGA GCA CGC CCT CCT CGC CGC 623,
226-244.sup.d TCG C 1985, 1986.sup.c 2107 5'-GCG AGC TCC CCA TCT
CTG GTT GGC ACG 623, 390-408.sup.d CTC GC 1985, 1986.sup.c
Bacterial genus: Legionella sp. 2083 5'-CCG CCG ATG TTC CGT AAA TTA
CTT GAI 111-112.sup.d 488-519.sup.e GAA GGT CGA GCC GGC GG
.sup.aUnderlined nucleotides indicate the molecular beacon's stem.
.sup.bThis sequence is from the complementary DNA strand of the
sequence of the originating fragment given in the Sequence Listing.
.sup.cThese sequences were aligned to derive the corresponding
primer. .sup.dThe nucleotide positions refer to the C. neoformans
tuf (EF-1) sequence fragment (SEQ ID NO. 623). .sup.eThe nucleotide
positions refer to the L. pneumophila tuf (EF-1) sequence fragment
(SEQ ID NO. 112). Originating DNA fragment SEQ ID Nucleotide SEQ ID
NO. Nucleotide sequence.sup.a NO. position Fungal genus: Candida
sp. 2108 5'-GCG GGC AAC TTC RTC AAG AAG GTT GGT 414, 52-80.sup.c
TAC AAC CCG CCC GC 417, 422, 424, 426, 429, 624.sup.b 2109 5'-GCGP
GGC CCA ATC TCT GGT TGG AAY GGT Same as 100-125.sup.c GAC AAG CCC
GC SEQ ID NO. 2108 Bacterial group: Pseudomonads 2121 5'-CGA CCG
CIA GCC GCA CAC CAA GTT CCG 153- 598-616.sup.e GTC G 155, 205, 880,
2137.sup.d, 2138.sup.d,b .sup.aUnderlined nucleotides indicate the
molecular beacon's stem. .sup.bThese sequences were aligned to
derive the corresponding primer. .sup.cThe nucleotide positions
refer to the C. albicans tuf (EF-1) sequence fragment (SEQ ID NO.
624). .sup.dSequence from databases. .sup.eThe nucleotide positions
refer to the P. aeruginosa tuf sequence fragment (SEQ ID NO.
153).
TABLE-US-00087 Annex XLVIII: Molecular beacon internal
hybridization probes for specific detection of ddl and mtl gene
sequences. Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.
Nucleotide sequence.sup.a NO. position Bacterial species: E.
faecium (ddl) 1237 5'-GCG AGC CGC GAA ATC GAA GTT GCT GTA
1242.sup.b 334-359 TTA GGG CTC GC Bacterial species: E. faecalis
(mtl) 1238 5'-GCG AGC GGC GTT AAT TTT GGC ACC GAA 1243.sup.b
631-656 GAA GAG CTC GC .sup.aUnderlined nucleotides indicate the
molecular beacon's stem. .sup.bSequence from databases.
TABLE-US-00088 Annex XLIX: Internal hybridization probe for
specific detection of S. aureus sequences of unknown coding
potential. Originating DNA fragment SEQ SEQ ID ID Nucleotide NO.
Nucleotide sequence NO. position Bacterial species: Staphylococcus
aureus 1234 5'-ACT AAA TAA ACG CTC ATT CG 1244 35-54
TABLE-US-00089 Annex L: Specific and ubiquitous primers for nucleic
acid amplification (antimicrobial agents resistance genes
sequences). Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.
Nucleotide sequence NO. position Resistance gene: aac(2')-Ia 1344
5'-AGC AGC AAC GAT GTT ACG CAG CAG 1348.sup.a 163-186 1345.sup.b
5'-CCC GCC GAG CAT TTC AAC TAT TG 1348.sup.a 392-414 1346 5'-GAT
GTT ACG CAG CAG GGC AGT C 1348.sup.a 172-193 1347.sup.b 5'-ACC AAG
CAG GTT CGC AGT CAA GTA 1348.sup.a 467-490 Resistance gene:
aac(3')-Ib 1349 5'-CAG CCG ACC AAT GAG TAT CTT GCC 1351.sup.a
178-201 1350.sup.b 5'-TAA TCA GGG CAG TTG CGA CTC CTA 1351.sup.a
356-379 Resistance gene: aac(3')-IIb 1352 5'-CCA CGC TGA CAG AGC
CGC ACC G 1356.sup.a 383-404 1353.sup.b 5'-GGC CAG CTC CCA TCG GAC
CCT G 1356.sup.a 585-606 1354 5'-CAC GCT GAC AGA GCC GCA CCG
1356.sup.a 384-404 1355.sup.b 5'-ATG CCG TTG CTG TCG AAA TCC TCG
1356.sup.a 606-629 Resistance gene: aac(3')-IVa 1357 5'-GCC CAT CCA
TTT GCC TTT GC 1361.sup.a 295-314 1358.sup.b 5'-GCG TAC CAA CTT GCC
ATC CTG AAG 1361.sup.a 517-540 1359 5'-TGC CCC TGC CAC CTC ACT C
1361.sup.a 356-374 1360.sup.b 5'-CGT ACC AAC TTG CCA TCC TGA AGA
1361.sup.a 516-539 Resistance gene: aac(3')-VIa 1362 5'-CGC CGC CAT
CGC CCA AAG CTG G 1366.sup.a 285-306 1363.sup.b 5'-CGG CAT AAT GGA
GCG CGG TGA CTG 1366.sup.a 551-574 1364 5'-TTT CTC GCC CAC GCA GGA
AAA ATC 1366.sup.a 502-525 1365.sup.b 5'-CAT CCT CGA CGA ATA TGC
CGC G 1366.sup.a 681-702 Resistance gene: aac(6')-Ia 1367 5'-CAA
ATA TAC TAA CAG AAG CGT TCA 1371.sup.a 56-79 1368.sup.b 5'-AGG ATC
TTG CCA ATA CCT TTA T 1371.sup.a 269-290 1379 5'-AAA CCT TTG TTT
CGG TCT GCT AAT 1371.sup.a 153-176 1380.sup.b 5'-AAG CGA TTC CAA
TAA TAC CTT GCT 1371.sup.a 320-343 Resistance gene: aac(6')-Ic 1372
5'-GCT TTC GTT GCC TTT GCC GAG GTC 1376.sup.a 157-180 1373.sup.b
5'-CAC CCC TGT TGC TTC GCC CAC TC 1376.sup.a 304-326 1374 5'-AGA
TAT TGG CTT CGC CGC ACC ACA 1376.sup.a 104-127 1375.sup.b 5'-CCC
TGT TGC TTC GCC CAC TCC TG 1376.sup.a 301-323 Resistance gene:
ant(3')-Ia 1377 5'-GCC GTG GGT CGA TGT TTG ATG TTA 1381.sup.a
100-123 1378.sup.b 5'-GCT CGA TGA CGC CAA CTA CCT CTG 1381.sup.a
221-244 1379 5'-AGC AGC AAC GAT GTT ACG CAG CAG 1381.sup.a 127-150
1380.sup.b 5'-CGC TCG ATG ACG CCA ACT ACC TCT 1381.sup.a 222-245
Resistance gene: ant(4')-Ia 1382 5'-TAG ATA TGA TAG GCG GTA AAA AGC
1386.sup.a 149-172 1383.sup.b 5'-CCC AAA TTC GAG TAA GAG GTA TT
1386.sup.a 386-408 1384 5'-GAT ATG ATA GGC GGT AAA AAG C 1386.sup.a
151-172 1385.sup.b 5'-TCC CAA ATT CGA GTA AGA GGT A 1386.sup.a
388-409 Resistance gene: aph(3')-Ia 1387 5'-TTA TGC CTC TTC CGA CCA
TCA AGC 1391.sup.a 233-256 1338.sup.b 5'-TAC GCT CGT CAT CAA AAT
CAC TCG 1391.sup.a 488-511 1389 5'-GAA TAA CGG TTT GGT TGA TGC GAG
1391.sup.a 468-491 1390.sup.b 5'-ATG GCA AGA TCC TGG TAT CGG TCT
1391.sup.a 669-692 Resistance gene: aph(3')-IIa 1392 5'-TGG GTG GAG
AGG CTA TTC GGC TAT 1396.sup.a 43-66 1393.sup.b 5'-CAG TCC CTT CCC
GCT TCA GTG AC 1396.sup.a 250-272 1394 5'-GAC GTT GTC ACT GAA GCG
GGA AGG 1396.sup.a 244-267 1395.sup.b 5'-CTT GGT GGT CGA ATG GGC
AGG TAG 1396.sup.a 386-409 Resistance gene: aph(3')-IIIa 1397
5'-GTG GGA GAA AAT GAA AAC CTA T 1401.sup.a 103-124 1398.sup.b
5'-ATG GAG TGA AAG AGC CTG AT 1401.sup.a 355-374 1399 5'-ACC TAT
GAT GTG GAA CGG GAA AAG 1401.sup.a 160-183 1400.sup.b 5'-CGA TGG
AGT GAA AGA GCC TGA TG 1401.sup.a 354-376 Resistance gene:
aph(3')-VIa 1402 5'-TAT TCA ACA ATT TAT CGG AAA CAG 1406.sup.a
18-41 1403.sup.b 5'-TCA GAG AGC CAA CTC AAC ATT TT 1406.sup.a
175-197 1404 5'-AAA CAG CGT TTT AGA GCC AAA TAA 1406.sup.a 36-59
1405.sup.b 5'-TTC TCA GAG AGC CAA CTC AAC ATT 1406.sup.a 177-200
Resistance gene: blaCARB 1407 5'-CCC TGT AAT AGA AAA GCA AGT AGG
1411.sup.a 351-374 1408.sup.b 5'-TTG TCG TAT CCC TCA AAT CAC C
1411.sup.a 556-577 1409 5'-TGG GAT TAC AAT GGC AAT CAG CG
1411.sup.a 205-227 1410.sup.b 5'-GGG GAA TAG GTC ACA AGA TCT GCT T
1411.sup.a 329-353 Resistance gene: blaCMY-2 1412 5'-GAG AAA ACG
CTC CAG CAG GGC 1416.sup.a 793-813 1413.sup.b 5'-CAT GAG GCT TTC
ACT GCG GGG 1416.sup.a 975-995 1414 5'-TAT CGT TAA TCG CAC CAT CAC
1416.sup.a 90-110 1415.sup.b 5'-ATG CAG TAA TGC GGC TTT ATC
1416.sup.a 439-459 Resistance genes: blaCTX-M-1, blaCTX-M-2 1417
5'-TGG TTA ACT AYA ATC CSA TTG CGG A 1423.sup.a 314-338 1418.sup.b
5'-ATG CTT TAC CCA GCG TCA GAT T 1423.sup.a 583-604 Resistance
gene: blaCTX-M-1 1419 5'-CGA TGA ATA AGC TGA TTT CTC ACG 1423.sup.a
410-433 1420.sup.b 5'-TGC TTT ACC CAG CGT CAG ATT ACG 1423.sup.a
580-603 1421 5'-AAT TAG AGC GGC AGT CGG GAG GAA 1423.sup.a 116-139
1422.sup.b 5'-GAA ATC AGC TTA TTC ATC GCC ACG 1423.sup.a 405-428
Resistance gene: b1aCTX-M-2 1424 5'-GTT AAC GGT GAT GGC GAC GCT AC
1428.sup.a 30-52 1425.sup.b 5'-GAA TTA TCG GCG GTG TTA ATC AGC
1428.sup.a 153-176 1426 5'-CAC GCT CAA TAC CGC CAT TCC A 1428.sup.a
510-531 1427.sup.b 5'-TTA TCG CCC ACT ACC CAT GAT TTC 1428.sup.a
687-710 Resistance gene: b1aIMP 1429 5'-TTT ACG GCT AAA GAT ACT GAA
AAG T 1433.sup.a 205-229 1430.sup.b 5'-GTT TAA TAA AAC AAC CAC CGA
ATA AT 1433.sup.a 513-538 1431 5'-TAA TTG ACA CTC CAT TTA CGG CTA A
1433.sup.a 191-215 1432.sup.b 5'-ACC GAA TAA TAT TTT CCT TTC AGG CA
1433.sup.a 497-522 Resistance gene: b1aOXA2 1434 5'-CAC AAT CAA GAC
CAA GAT TTG CGA T 1438.sup.a 319-343 1435.sup.b 5'-GAA AGG GCA GCT
CGT TAC GAT AGA G 1438.sup.a 532-556 Resistance gene: b1aOXA10 1436
5'-CAG CAT CAA CAT TTA AGA TCC CCA 1439.sup.a 194-217 1437.sup.b
5'-CTC CAC TTG ATT AAC TGC GGA AAT TC 1439.sup.a 479-504 Resistance
gene: b1aPER-1 1440 5'-AGA CCG TTA TCG TAA ACA GGG CTA AG
1442.sup.a 281-306 1441.sup.b 5'-TTT TTT GCT CAA ACT TTT TCA GGA TC
1442.sup.a 579-604 Resistance gene: b1aPER-2 1443 5'-CTT CTG CTC
TGC TGA TGC TTG GC 1445.sup.a 32-54 1444.sup.b 5'-GGC GAC CAG GTA
TTT TGT AAT ACT GC 1445.sup.a 304-329 Resistance genes: b1aPER-1,
b1aPER-2 1446 5'-GGC CTG YGA TTT GTT ATT TGA ACT GGT 1442.sup.a
414-440 1447.sup.b 5'-CGC TST GGT CCT GTG GTG GTT TC 1442.sup.a
652-674 1448 5'-GAT CAG GTG CAR TAT CAA AAC TGG AC 1442.sup.a
532-557 1449.sup.b 5'-AGC WGG TAA CAA YCC TTT TAA CCG CT 1442.sup.a
671-696 Resistance gene: b1aSHV 1883 5'-AGC CGC TTG AGC AAA TTA AAC
TA 1900.sup.a 71-93 1884.sup.b 5'-GTA TCC CGC AGA TAA ATC ACC AC
1900.sup.a 763-785 1885 5'-AGC GAA AAA CAC CTT GCC GAC 1900.sup.a
313-333 1884.sup.b 5'-GTA TCC CGC AGA TAA ATC ACC AC 1900.sup.a
763-785 Resistance gene: b1aTEM 1906 5'-CCT TAT TCC CTT TTT TGC GG
1927.sup.a 27-46 1907.sup.b 5'-CAC CTA TCT CAG CGA TCT GTC T
1927.sup.a 817-838 1908 5'-AAC AGC GGT AAG ATC CTT GAG AG
1927.sup.a 148-170 1907.sup.b 5'-CAC CTA TCT CAG CGA TCT GTC T
1927.sup.a 817-838 Resistance gene: catI 2145 5'-GCA AGA TGT GGC
GTG TTA CGG T 2147.sup.a 363-384 2146.sup.b 5'-GGG GCG AAG AAG TTG
TCC ATA TT 2147.sup.a 484-506 Resistance gene: catII 2148 5'-CAG
ATT AAA TGC GGA TTC AGC C 2150.sup.a 67-88 2149.sup.b 5'-ATC AGG
TAA ATC ATC AGC GGA TA 2150.sup.a 151-173 Resistance gene: catIII
2151 5'-ATA TTT CAG CAT TAC CTT GGG TT 2153.sup.a 419-441
2152.sup.b 5'-TAC ACA ACT CTT GTA GCC GAT TA 2153.sup.a 603-625
Resistance gene: catP 2154 5'-CGC CAT TCA GAG TTT AGG AC 2156.sup.a
178-197 2155.sup.b 5'-TTC CAT ACC GTT GCG TAT CAC TT 2156.sup.a
339-361 Resistance gene: cat 2157 5'-CCA CAG AAA TTG ATA TTA GTG
TTT TAT 2159.sup.a 89-115 2158.sup.b 5'-TCG CTA TTG TAA CCA GTT CTA
2159.sup.a 201-221 2160 5'-TTT TGA ACA CTA TTT TAA CCA GC
2162.sup.a 48-70 2161.sup.b 5'-GAT TTA ACT TAT CCC AAT AAC CT
2162.sup.a 231-253 Resistance gene: dfrA 1450 5'-ACC ACT GGG AAT
ACA CTT GTA ATG GC 1452.sup.a 106-131 1451.sup.b 5'-ATC TAC CTG GTC
AAT CAT TGC TTC GT 1452.sup.a 296-321 Resistance gene: dhfrIa 1457
5'-CAA AGG TGA ACA GCT CCT GTT T 1461.sup.a 75-96 1458.sup.b 5'-TCC
GTT ATT TTC TTT AGG TTG GTT AAA 1461.sup.a 249-275 1459 5'-AAG GTG
AAC AGC TCC TGT TT 1461.sup.a 77-96 1560.sup.b 5'-GAT CAC TAC GTT
CTC ATT GTC A 1461.sup.a 207-228 Resistance genes: dhfrIa, dhfrXV
1453 5'-ATC GAA GAA TGG AGT TAT CGG RAA TG 1461.sup.a 27-52
1454.sup.b 5'-CCT AAA AYT RCT GGG GAT TTC WGG A 1461.sup.a 384-408
1455 5'-CAG GTG GTG GGG AGA TAT ACA AAA 1461.sup.a 290-313
1456.sup.b 5'-TAT GTT AGA SRC GAA GTC TTG GKT AA 1461.sup.a 416-441
Resistance gene: dhfrIb 1466 5'-AAG CAT TGA CCT ACA ATC AGT GT
1470.sup.a 98-120 1467.sup.b 5'-AAT ACA ACT ACA TTG TCA TCA TTT GAT
1470.sup.a 204-230 1468 5'-CGT TAC CCG CTC AGG TTG GAC ATC AA
1470.sup.a 183-208 1469.sup.b 5'-CAT CCC CCT CTG GCT CGA TGT CG
1470.sup.a 354-376 Resistance gene: dhfrV 1471 5'-GAT AAT GAC AAC
GTA ATA GTA TTC CC 1475.sup.a 208-233 1472.sup.b 5'-GCT CAA TAT CAA
TCG TCG ATA TA 1475.sup.a 342-364 1473 5'-TTA AAG CCT TGA CGT ACA
ACC AGT GG 1475.sup.a 95-120 1474.sup.b 5'-TGG GCA ATG TTT CTC TGT
AAA TCT CC 1475.sup.a 300-325 Resistance genes: dhfrIb, dhfrV 1462
5'-GCA CTC CCY AAT AGG AAA TAC GC 1470.sup.a 157-179 1463.sup.b
5'-AGT GTT GCT CAA AAA CAA CTT CG 1470.sup.a 405-427 1464 5'-ACG
TTY GAA TCT ATG GGM GCA CT 1470.sup.a 139-161 1465.sup.b 5'-GTC GAT
AAG TGG AGC GTA GAG GC 1470.sup.a 328-350 Resistance gene: dhfrVI
1476 5'-GGC GAG CAG CTC CTA TTC AAA G 1480.sup.a 79-100 1477.sup.b
5'-TAG GTA AGC TAA TGC CGA TTC AAC A 1480.sup.a 237-261 1478 5'-GAG
AAT GGA GTA ATT GGC TCT GGA TT 1480.sup.a 31-56 1479.sup.b 5'-GCG
AAA TAC ACA ACA TCA GGG TCA T 1480.sup.a 209-233 Resistance gene:
dhfrVII 1485 5'-AAA ATG GCG TAA TCG GTA ATG GC 1489.sup.a 32-54
1486.sup.b 5'-CAT TTG AGC TTG AAA TTC CTT TCC TC 1489.sup.a 189-214
1487 5'-AAT CGA AAA TAT GCA GTA GTG TCG AG 1489.sup.a 166-191
1488.sup.b 5'-AGA CTA TTG TAG ATT TGA CCG CCA 1489.sup.a 294-317
Resistance genes: dhfrVII, dhfrXVII
1481 5'-RTT ACA GAT CAT KTA TAT GTC TCT 1489.sup.a 268-291
1482.sup.b 5'-TAA TTT ATA TTA GAC AWA AAA AAC TG 1489.sup.a 421-446
1483 5'-CAR YGT CAG AAA ATG GCG TAA TC 1489.sup.a 23-45 1484.sup.b
5'-TKC AAA GCR WTT TCT ATT GAA GGA AA 1489.sup.a 229-254 Resistance
gene: dhfrVIII 1490 5'-GAC CTA TGA GAG CTT GCC CGT CAA A 1494.sup.a
144-168 1491.sup.b 5'-TCG CCT TCG TAC AGT CGC TTA ACA AA 1494.sup.a
376-401 1492 5'-CAT TTT AGC TGC CAC CGC CAA TGG TT 1494.sup.a 18-43
1493.sup.b 5'-GCG TCG CTG ACG TTG TTC ACG AAG A 1494.sup.a 245-269
Resistance gene: dhfrIX 1495 5'-TCT CTA AAC ATG ATT GTC GCT GTC
1499.sup.a 7-30 1496.sup.b 5'-CAG TGA GGC AAA AGT TTT TCT ACC
1499.sup.a 133-156 1497 5'-CGG ACG ACT TCA TGT GGT AGT CAG T
1499.sup.a 171-195 1498.sup.b 5'-TTT GTT TTC AGT AAT GGT CGG GAC CT
1499.sup.a 446-471 Resistance gene: dhfrXII 1500 5'-ATC GGG TTA TTG
GCA ATG GTC CTA 1504.sup.a 50-73 1501.sup.b 5'-GCG GTA GTT AGC TTG
GCG TGA GAT T 1504.sup.a 201-225 1502 5'-GCG GGC GGA GCT GAG ATA
TAC A 1504.sup.a 304-325 1503.sup.b 5'-AAC GGA GTG GGT GTA CGG AAT
TAC AG 1504.sup.a 452-477 Resistance gene: dhfrXIII 1505 5'-ATT TTT
CGC AGG CTC ACC GAG AGC 1507.sup.a 106-129 1506.sup.b 5'-CGG ATG
AGA CAA CCT CGA ATT CTG CTG 1507.sup.a 413-439 Resistance gene:
dhfrXV 1508 5'-AGA ATG TAT TGG TAT TTC CAT CTA TCG 1512.sup.a
215-241 1509.sup.b 5'-CAA TGT CGA TTG TTG AAA TAT GTA AA 1512.sup.a
336-361 1510 5'-TGG AGT GCC AAA GGG GAA CAA T 1512.sup.a 67-88
1511.sup.b 5'-CAG ACA CAA TCA CAT GAT CCG TTA TCG 1512.sup.a
266-292 Resistance gene: dhfrXVII 1513 5'-TTC AAG CTC AAA TGA AAA
CGT CC 1517.sup.a 201-223 1514.sup.b 5'-GAA ATT CTC AGG CAT TAT AGG
GAA T 1517.sup.a 381-405 1515 5'-GTG GTC AGT AAA AGG TGA GCA AC
1517.sup.a 66-88 1516.sup.b 5'-TCT TTC AAA GCA TTT TCT ATT GAA GG
1517.sup.a 232-257 Resistance gene: embB 2102 5'-CAC CTT CAC CCT
GAC CGA CG 2105.sup.a 822-841 2103.sup.b 5'-CGA ACC AGC GGA AAT AGT
TGG AC 2105.sup.a 948-970 Resistance genes: ereA, ereA2 1528 5'-AAC
TTG AGC GAT TTT CGG ATA CCC TG 1530.sup.a 80-105 1529.sup.b 5'-TTG
CCG ATG AAA TAA CCG CCG ACT 1530.sup.a 317-340 Resistance gene:
ereB 1531 5'-TCT TTT TGT TAC GAC ATA CGC TTT T 1535.sup.a 152-176
1532.sup.b 5'-AGT GCT TCT TTA TCC GCT GTT CTA 1535.sup.a 456-479
1533 5'-CAG CGG ATA AAG AAG CAC TAC ACA TT 1535.sup.a 461-486
1534.sup.b 5'-CCT CCT GAA ATA AAG CCC GAC AT 1535.sup.a 727-749
Resistance gene: gyrA 1340 5'-GAA CAA GGT ATG ACA CCG GAT AAA T
1299.sup.a 163-188 1341.sup.b 5'-GAT AAC TGA AAT CCT GAG CCA TAC G
1299.sup.a 274-299 1936 5'-TAC CAC CCG CAC GGC 1954.sup.a 205-219
1937.sup.b 5'-CGG AGT CGC CGT CGA TG 1954.sup.a 309-325 1942 5'-GAC
TGG AAC AAA GCC TAT AAA AAA TCA 1954.sup.a 148-174 1937.sup.b
5'-CGG AGT CGC CGT CGA TG 1954.sup.a 309-325 2040 5'-TGT GAC CCC
AGA CAA ACC C 2054.sup.a 33-51 2041.sup.b 5'-GTT GAG CGG CAG CAC
TAT CT 2054.sup.a 207-226 Resistance gene: inhA 2098 5'-CTG AGT CAC
ACC GAC AAA CGT C 2101.sup.a 910-931 2099.sup.b 5'-CCA GGA CTG AAC
GGG ATA CGA A 2101.sup.a 1074-1095 Resistance genes: linA, linA'
1536 5'-AGA TGT ATT AAC TGG AAA ACA ACA A 1540.sup.a 99-123
1537.sup.b 5'-CTT TGT AAT TAG TTT CTG AAA ACC A 1540.sup.a 352-376
1538 5'-TTA GAA GAT ATA GGA TAC AAA ATA GAA G 1540.sup.a 187-214
1539.sup.b 5'-GAA TGA AAA AGA AGT TGA GCT T 1540.sup.a 404-425
Resistance gene: linB 1541 5'-TGA TAA TCT TAT ACG TGG GGA ATT T
1545.sup.a 246-270 1542.sup.b 5'-ATA ATT TTC TAA TTG CCC TGT TTC AT
1545.sup.a 359-384 1543 5'-GGG CAA TTA GAA AAT TAT TTA TCA GA
1545.sup.a 367-392 1544.sup.b 5'-TTT TAC TCA TGT TTA GCC AAT TAT CA
1545.sup.a 579-604 Resistance gene: mefA 1546 5'-CAA GAA GGA ATG
GCT GTA CTA C 1548.sup.a 625-646 1547.sup.b 5'-TAA TTC CCA AAT AAC
CCT AAT AAT AGA 1548.sup.a 816-842 Resistance gene: mefE 1549
5'-GCT TAT TAT TAG GAA GAT TAG GGG GC 1551.sup.a 815-840 1550.sup.b
5'-TAG CAA GTG ACA TGA TAC TTC CGA 1551.sup.a 1052-1075 Resistance
genes: mefA, mefE 1552 5'-GGC AAG CAG TAT CAT TAA TCA CTA
1548.sup.a 50-73 1553.sup.b 5'-CAA TGC TAC GGA TAA ACA ATA CTA TC
1548.sup.a 318-343 1554 5'-AGA AAA TTA AGC CTG AAT ATT TAG GAC
1548.sup.a 1010-1035 1555.sup.b 5'-TAG TAA AAA CCA ATG ATT TAC ACC
G 1548.sup.a 1119-1143 Resistance genes: mphA, mphK 1556 5'-ACT GTA
CGC ACT TGC AGC CCG ACA T 1560.sup.a 33-57 1557.sup.b 5'-GAA CGG
CAG GCG ATT CTT GAG CAT 1560.sup.a 214-237 1558 5'-GTG GTG GTG CAT
GGC GAT CTC T 1560.sup.a 583-604 1559.sup.b 5'-GCC GCA GCG AGG TAC
TCT TCG TTA 1560.sup.a 855-878 Resistance gene: mupA 2142 5'-GCC
TTA ATT TCG GAT AGT GC 2144.sup.a 1831-1850 2143.sup.b 5'-GAG AAA
GAG CCC AAT TAT CTA ATG T 2144.sup.a 2002-2026 Resistance gene:
parC 1342 5'-GAT GTT ATT GGT CAA TAT CAT CCA 1321.sup.a 205-229
1343.sup.b 5'-AAG AAA CTG TCT CTT TAT TAA TAT CAC GT 1321.sup.a
396-425 1934 5'-GAA CGC CAG CGC GAA ATT CAA AAA G 1781 67-91
1935.sup.b 5'-AGC TCG GCA TAC TTC GAC AGG 1781 277-297 2044 5'-ACC
GTA AGT CGG CCA AGT CA 2055.sup.a 176-195 2045.sup.b 5'-GTT CTT TCT
CCG TAT CGT C 2055.sup.a 436-454 Resistance gene: ppflo-like 2163
5'-ACC TTC ATC CTA CCG ATG TGG GTT 2165.sup.a 922-945 2164.sup.b
5'-CAA CGA CAC CAG CAC TGC CAT TG 2165.sup.a 1136-1158 Resistance
gene: rpoB 2065 5'-CCA GGA CGT GGA GGC GAT CAC A 2072.sup.a
1218-1239 2066.sup.b 5'-CAC CGA CAG CGA GCC GAT CAG A 2072.sup.a
1485-1506 Resistance gene: satG 1581 5'-AAT TGG GGA CTA CAC CTA TTA
TGA TG 1585.sup.a 93-118 1582.sup.b 5'-GGC AAA TCA GTC AGT TCA GGA
GT 1585.sup.a 310-332 1583 5'-CGA TTG GCA ACA ATA CAC TCC TG
1585.sup.a 294-316 1584.sup.b 5'-TCA CCT ATT TTT ACG CCT GGT AGG AC
1585.sup.a 388-413 Resistance gene: sulII 1961 5'-GCT CAA GGC AGA
TGG CAT TCC C 1965.sup.a 222-243 1962.sup.b 5'-GGA CAA GGC GGT TGC
GTT TGA T 1965.sup.a 496-517 1963 5'-CAT TCC CGT CTC GCT CGA CAG T
1965.sup.a 237-258 1964.sup.b 5'-ATC TGC CTG CCC GTC TTG C
1965.sup.a 393-411 Resistance gene: tetB 1966 5'-CAT GCC AGT CTT
GCC AAC G 1970.sup.a 66-84 1967.sup.b 5'-CAG CAA TAA GTA ATC CAG
CGA TG 1970.sup.a 242-264 1968 5'-GGA GAG ATT TCA CCG CAT AG
1970.sup.a 457-476 1969.sup.b 5'-AGC CAA CCA TCA TGC TAT TCC A
1970.sup.a 721-742 Resistance gene: tetM 1586 5'-ATT CCC ACA ATC
TTT TTT ATC AAT AA 1590.sup.a 361-386 1587.sup.b 5'-CAT TGT TCA GAT
TCG GTA AAG TTC 1590.sup.a 501-524 1588 5'-GTT TTT GAA GTT AAA TAG
TGT TCT T 1590.sup.a 957-981 1589.sup.b 5'-CTT CCA TTT GTA CTT TCC
CTA 1590.sup.a 1172-1192 Resistance gene: vatB 1609 5'-GCC CTG ATC
CAA ATA GCA TAT A 1613.sup.a 11-32 1610.sup.b 5'-CCT GGC ATA ACA
GTA ACA TTC TG 1613.sup.a 379-401 1611 5'-TGG GAA AAA GCA ACT CCA
TCT C 1613.sup.a 301-322 1612.sup.b 5'-ACA ACT GAA TTC GCA GCA ACA
AT 1613.sup.a 424-446 Resistance gene: vatC 1614 5'-CCA ATC CAG AAG
AAA TAT ACC C 1618.sup.a 26-47 1615.sup.b 5'-ATT AGT TTA TCC CCA
ATC AAT TCA 1618.sup.a 177-200 1616 5'-ATA ATG AAT GGG GCT AAT CAT
CGT AT 1618.sup.a 241-266 1617.sup.b 5'-GCC AAC AAC TGA ATA AGG ATC
AAC 1618.sup.a 463-486 Resistance gene: vga 1619 5'-AAG GCA AAA TAA
AAG GAG CAA AGC 1623.sup.a 641-664 1620.sup.b 5'-TGT ACC CGA GAC
ATC TTC ACC AC 1623.sup.a 821-843 1621 5'-AAT TGA AGG ACG GGT ATT
GTG GAA AG 1623.sup.a 843-868 1622.sup.b 5'-CGA TTT TGA CAG ATG GCG
ATA ATG AA 1623.sup.a 975-1000 Resistance gene: vgaB 1624 5'-TTC
TTT AAT GCT CGT AGA TGA ACC TA 1628.sup.a 354-379 1625.sup.b 5'-TTT
TCG TAT TCT TCT TGT TGC TTT C 1628.sup.a 578-602 1626 5'-AGG AAT
GAT TAA GCC CCC TTC AAA AA 1628.sup.a 663-688 1627.sup.b 5'-TTA CAT
TGC GAC CAT GAA ATT GCT CT 1628.sup.a 849-874 Resistance genes:
vgb, vgh 1629 5'-AAG GGG AAA GTT TGG ATT ACA CAA CA 1633.sup.a
73-98 1630.sup.b 5'-GAA CCA CAG GGC ATT ATC AGA ACC 1633.sup.a
445-468 1631 5'-CGA CGA TGC TTT ATG GTT TGT 1633.sup.a 576-596
1632.sup.b 5'-GTT AAT TTG CCT ATC TTG TCA CAC TC 1633.sup.a 850-875
Resistance gene: vgbB 1634 5'-TTA ACT TGT CTA TTC CCG ATT CAG G
1882.sup.a 23-47 1635.sup.b 5'-GCT GTG GCA ATG GAT ATT CTG TA
1882.sup.a 267-289 1636 5'-TTC CTA CCC CTG ATG CTA AAG TGA
1882.sup.a 155-178 1637.sup.b 5'-CAA AGT GCG TTA TCC GAA CCT AA
1882.sup.a 442-464 Sequencing primers Resistance gene: gyrA 1290
5'-GAY TAY GCI ATG ISI GTI ATH GT 1299.sup.a 70-83 1292.sup.b
5'-ARI SCY TCI ARI ATR TGI GC 1299.sup.a 1132-1152 1291 5'-GCI YTI
CCI GAY GTI MGI GAY GG 1299.sup.a 100-123 1292.sup.b 5'-ARI SCY TCI
ARI ATR TGI GC 1299.sup.a 1132-1152 1293 5'-ATG GCT GAA TTA CCT CAA
TC 1299.sup.a 1-21 1294.sup.b 5'-ATG ATT GTT GTA TAT CTT CTT CAA C
1299.sup.a 2626-2651 1295.sup.b 5'-CAG AAA GTT TGA AGC GTT GT
1299.sup.a 1255-1275 1296 5'-AAC GAT TCG TGA GTC AGA TA 1299.sup.a
1188-1208 1297 5'-CGG TCA ACA TTG AGG AAG AGC T 1300.sup.a 29-51
1298.sup.b 5'-ACG AAA TCG ACC GTC TCT TTT TC 1300.sup.a 415-437
Resistance gene: gyrB 1301 5'-GTI MGI AWI MGI CCI GSI ATG TA
1307.sup.a 82-105 1302.sup.b 5'-TAI ADI GGI GGI KKI GCI ATR TA
1307.sup.a 1600-1623 1303 5'-GGI GAI GAI DYI MGI GAR GG 1307.sup.a
955-975 1304.sup.b 5'-CIA RYT TIK YIT TIG TYT G 1307.sup.a
1024-1043 1305 5'-ATG GTG ACT GCA TTG TCA GAT G 1307.sup.a 1-23
1306.sup.b 5'-GTC TAC GGT TTT CTA CAA CGT C 1307.sup.a 1858-1888
Resistance gene: parC 1308 5'-ATG TAY GTI ATI ATG GAY MGI GC
1320.sup.a 67-90 1309.sup.b 5'-ATI ATY TTR TTI CCY TTI CCY TT
1320.sup.a 1993-2016 1310 5'-ATI ATI TSI ATI ACY TCR TC 1320.sup.a
1112-1132 1311.sup.b 5'-GAR ATG AAR ATI MGI GGI GAR CA 1320.sup.a
1288-1311 1312 5'-AAR TAY ATI ATI CAR GAR MGI GC 1321.sup.a 67-90
1313.sup.b 5'-AMI AYI CKR TGI GGI TTI TTY TT 1321.sup.a 2212-2235
1314 5'-TAI GAI TTY ACI GAI SMI CAR GC 1321.sup.a 1228-1251
1315.sup.b 5'-ACI ATI GCI TCI GCY TGI KSY TC 1321.sup.a 1240-1263
1316 5'-GTG AGT GAA ATA ATT CAA GAT T 1321.sup.a 1-23 1317.sup.b
5'-CAC CAA AAT CAT CTG TAT CTA C 1321.sup.a 2356-2378
1318 5'-ACC TAY TCS ATG TAC GTR ATC ATG GA 1320.sup.a 58-84
1319.sup.b 5'-AGR TCG TCI ACC ATC GGY AGY TT 1320.sup.a 832-855
Resistance gene: parE 1322 5'-RTI GAI AAY ISI GTI GAY GAR G
1328.sup.a 133-155 1325.sup.b 5'-RTT CAT YTC ICC IAR ICC YTT
1328.sup.a 1732-1752 1323 5'-ACI AWR SAI GGI GGI ACI CAY G
1328.sup.a 829-850 1324.sup.b 5'-CCI CCI GCI SWR TCI CCY TC
1328.sup.a 1280-1302 1326 5'-TGA TTC AAT ACA GGT TTT AGA G
1328.sup.a 27-49 1327.sup.b 5'-CTA GAT TTC CTC CTC ATC AAA T
1328.sup.a 1971-1993 .sup.aSequence from databases. .sup.bThese
sequences are from the complementary DNA strand of the sequence of
the originating fragment given in the Sequence Listing.
TABLE-US-00090 Annex LI: Internal hybridization probes for specific
detection of antimicrobial agents resistance genes sequences.
Originating DNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide
sequence NO. position Resistance gene: aph3'VIa 2252 5'-CCA CAT ACA
GTG TCT CTC 1406.sup.a 149-166 Resistance gene: blaSHV 1886 5'-GAC
GCC CGC GCC ACC ACT 1900.sup.a 484-501 1887 5'-GAC GCC CGC GAC ACC
ACT A 1899.sup.a 514-532 1888 5'-GAC GCC CGC AAC ACC ACT A
1901.sup.a 514-532 1889 5'-GTT CGC AAC TGC AGC TGC TG 1899.sup.a
593-612 1890 5'-TTC GCA ACG GCA GCT GCT G 1899.sup.a 594-612 1891
5'-CCG GAG CTG CCG AIC GGG 1902.sup.a 692-709 1892 5'-CGG AGC TGC
CAA RCG GGG 1903.sup.a 693-710 1893 5'-GGA GCT GGC GAR CGG GGT
1899.sup.a 694-711 1894 5'-GAC CGG AGC TAG CGA RCG 1904.sup.a
690-707 1895 5'-CGG AGC TAG CAA RCG GGG T 1905.sup.a 693-711 1896
5'-GAA ACG GAA CTG AAT GAG GCG 1899.sup.a 484-504 1897 5'-CAT TAC
CAT GGG CGA TAA CAG 1899.sup.a 366-386 1898 5'-CCA TTA CCA TGA GCG
ATA ACAG 1899.sup.a 365-386 Resistance gene: blaTEM 1909 5'-ATG ACT
TGG TTA AGT ACT CAC C 1928.sup.a 293-314 1910 5'-ATG ACT TGG TTG
AGT ACT CAC C 1927.sup.a 293-314 1911 5'-CCA TAA CCA TGG GTG ATA
ACA C 1928.sup.a 371-392 1912 5'-CCA TAA CCA TGA GTG ATA ACA C
1927.sup.a 371-392 1913 5'-CGC CTT GAT CAT TGG GAA CC 1928.sup.a
475-494 1914 5'-CGC CTT GAT CGT TGG GAA CC 1927.sup.a 475-494 1915
5'-CGC CTT GAT AGT TGG GAA CC 1929.sup.a 475-494 1916 5'-CGT GGG
TCT TGC GGT ATC AT 1927.sup.a 712-731 1917 5'-CGT GGG TCT GGC GGT
ATC AT 1930.sup.a 712-731 1918 5'-GTG GGT CTC ACG GTA TCA TTG
1927.sup.a 713-733 1919 5'-CGT GGG TCT CTC GGT ATC ATT 1931.sup.a
712-732 1920 5'-CGT GGI TCT CGC GGT ATC AT 1927.sup.a 712-731 1921
5'-CGT GGG TCT AGC GGT ATC ATT 1932.sup.a 713-733 1922 5'-GTT TTC
CAA TGA TTA GCA CTT TTA 1927.sup.a 188-211 1923 5'-GTT TTC CAA TGA
TAA GCA CTT TTA 1927.sup.a 188-211 1924 5'-GTT TTC CAA TGC TGA GCA
CTT TT 1932.sup.a 188-210 1925 5'-CGT TTT CCA ATG ATG AGC ACT TT
1927.sup.a 187-209 1926 5'-GTT TTC CAA TGG TGA GCA CTT TT
1933.sup.a 188-210 2006 5'-TGG AGC CGG TGA GCG TGG 1927.sup.a
699-716 2007 5'-TGG AGC CAG TGA GCG TGG 2010.sup.a 699-716 2008
5'-TCT GGA GCC GAT GAG CGT G 1929.sup.a 697-715 2009 5'-CTG GAG CCA
GTA AGC GTG G 2011.sup.a 698-716 2141 5'-CAC CAG TCA CAG AAA AGC
1927.sup.a 311-328 Resistance gene: dhfrIa 2253 5'-CAT TAC CCA ACC
GAA AGT A 1461.sup.a 158-176 Resistance gene: embB 2104 5'-CTG GGC
ATG GCI CGA GTC 2105.sup.a 910-927 Resistance gene: gyrA 1333
5'-TCA TGG TGA CTT ATC TAT TTA TG 1299.sup.a 240-263 1334 5'-CAT
CTA TTT ATA AAG CAA TGG TA 1299.sup.a 251-274 1335 5'-CTA TTT ATG
GAG CAA TGG T 1299.sup.a 254-273 1940 5'-GTA TCG TTG GTG ACG TAA T
1299.sup.a 206-224 1943 5'-GCT GGT GGA CGG CCA G 1954.sup.a 279-294
1945 5'-CGG CGA CTA CGC GGT AT 1954.sup.a 216-232 1946 5'-CGG CGA
CTT CGC GGT AT 1954.sup.a 216-232 1947 5'-CGG TAT ACG GCA CCA TCG T
1954.sup.a 227-245 1948 5'-GCG GTA TAC AAC ACC ATC G 1954.sup.a
226-244 1949 5'-CGG TAT ACG CCA CCA TCG T 1954.sup.a 227-245 2042
5'-CAC GGG GAT TTC TCT ATT TA 2054.sup.a 103-122 2043 5'-CAC GGG
GAT TAC TCT ATT TA 2054.sup.a 103-122 Resistance gene: inhA 2100
5'-GCG AGA CGA TAG GTT GTC 2101.sup.a 1017-1034 Resistance gene:
parC 1336 5'-TGG AGA CTA CTC AGT GT 1321.sup.a 232-249 1337 5'-TGG
AGA CTT CTC AGT GT 1321.sup.a 232-249 1338 5'-GTG TAC GGA GCA ATG
1321.sup.a 245-260 1339 5'-CCA GCG GAA ATG CGT 1321.sup.a 342-357
1941 5'-GCA ATG GTC CGT TTA AGT 1321.sup.a 253-270 1944 5'-TTT CGC
CGC CAT GCG TTA C 1781 247-265 1950 5'-GGC GAC ATC GCC TGC 1781
137-151 1951 5'-GGC GAC AGA GCC TGC TA 1781 137-153 1952 5'-CCT GCT
ATG GAG CGA TGG T 1781 147-165 1953 5'-CGC CTG CTA TAA AGC GAT GGT
1781 145-165 2046 5'-ACG GGG ATT TTT CTA TCT AT 2055.sup.a 227-246
Resistance gene: rpoB 2067 5'-AGC TGA GCC AAT TCA TGG 2072.sup.a
1304-1321 2068 5'-ATT CAT GGA CCA GAA CAA C 2072.sup.a 1314-1332
2069 5'-CGC TGT CGG GGT TGA CCC 2072.sup.a 1334-1351 2070 5'-GTT
GAC CCA CAA GCG CCG 2072.sup.a 1344-1361 2071 5'-CGA CTG TCG GCG
CTG GGG 2072.sup.a 1360-1377 Resistance gene: tetM 2254 5'-ACC TGA
ACA GAG AGA AAT G 1590.sup.a 1062-1080 .sup.aSequence from
databases.
TABLE-US-00091 Annex LII: Molecular beacon internal hybridization
probes for specific detection of atpD sequences. Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence.sup.a NO.
position Bacterial species: Bacteroides fragilis 2136 5'-CCA ACG
CGT CCT CAA TCA TTT CTA ACT TCT 929 353-382 ATG GCC GGC GTT GG
Bacterial species: Bordetella pertussis 2182 5'-GCG CGC CAA CGA CTT
CTA CCA CGA AAT GGA 1672 576-605 AGA GTC GCG CGC Bacterial group:
Campylobacter jejuni and C. coli 2133 5'-CCA CGC ACA WAA ACT TGT
TTT AGA AGT 1576, 44-73.sup.d AGC AGC WCA GCG TGG 1600, 1849, 1863,
2139.sup.b,c Fungal species: Candida glabrata 2078 5'-CCG AGC CTT
GGT CTT CGG CCA AAT GAA CGC 463 442-463 TCG G Fungal species:
Candida krusei 2075 5'-CCG AGC CAG GTT CTG AAG TCT CTG CAT TAT 468
720-748 TAG GTG CTC GG Fungal species: Candida lusitaniae 2080
5'-CCG AGC CGA AGA GGG CCA AGA TGT CGC TCG G 470 520-538 Fungal
species: Candida parapsilosis 2079 5'-CCG AGC GTT CAG TTA CTT CAG
TCC AAG CCG 472 837-860 GCT CGG Fungal species: Candida tropicalis
2077 5'-CCG AGC AAC CGA TCC AGC TCC AGC TAC GCT 475 877-897 CGG
Bacterial species: Klebsiella pneumoniae 2281 5'-CCC CCA GCT GGG
CGG CGG TAT CGA TGG GGG 317 40-59 .sup.aUnderlined nucleotides
indicate the molecular beacon's stem. .sup.bSequence from
databases. .sup.cThese sequences were aligned to derive the
corresponding primer. .sup.dThe nucleotide positions refer to the
C. jejuni atpD sequence fragment (SEQ ID NO. 1576). Originating DNA
fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence.sup.a NO.
position Fungal genus: Candida sp. 2076 5'-CCG AGC YGA YAA CAT TTT
CAG ATT CAC CCA 460-478, 697-723.sup.c RGC GCT CGG 663.sup.b
.sup.aUnderlined nucleotides indicate the molecular beacon's stem.
.sup.bThese sequences were aligned to derive the corresponding
primer. .sup.cThe nucleotide positions refer to the C. albicans
atpD sequence fragment (SEQ ID NO. 460).
TABLE-US-00092 Annex LIII: Internal hybridization probes for
specific detection of atpD sequences. Originating DNA fragment SEQ
SEQ ID Nucleotide ID NO. Nucleotide sequence NO. position Bacterial
species: Acinetobacter baumannii 2169 5'-CCC GTT TGC GAA AGG TGG
243 304-321 Bacterial species: Klebsiella pneumoniae 2167 5'-CAG
CAG CTG GGC GGC GGT 317 36-53
TABLE-US-00093 Annex LIV: Internal hybridization probes for
specific detection of ddl and mtl sequences. Originating DNA
fragment Nucleo- SEQ SEQ ID tide ID NO. Nucleotide sequence NO.
position Bacterial species: Enterococcus faecium (ddl) 2286 5'-AGT
TGC TGT ATT AGG AAA TG 2288.sup.a 784-803 2287 5'-TCG AAG TTG CTG
TAT TAG GA 2288.sup.a 780-799 Bacterial species: Enterococcus
faecalis mtl) 2289 5'-CAC CGA AGA AGA TGA AAA AA 1243.sup.a 264-283
2290 5'-TGG CAC CGA AGA AGA TGA 1243.sup.a 261-278 2291 5'-ATT TTG
GCA CCG AAG AAG A 1243.sup.a 257-275 .sup.aSequence from databases.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20100267012A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20100267012A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References