U.S. patent application number 09/368089 was filed with the patent office on 2001-08-02 for pna probes, probe sets, methods and kits pertaining to the universal detection of bacteria and eucarya.
This patent application is currently assigned to BOSTON PROBES INC.. Invention is credited to HYLDIG-NIELSEN, JENS J., O'KEEFE, HEATHER P..
Application Number | 20010010910 09/368089 |
Document ID | / |
Family ID | 26790415 |
Filed Date | 2001-08-02 |
United States Patent
Application |
20010010910 |
Kind Code |
A1 |
HYLDIG-NIELSEN, JENS J. ; et
al. |
August 2, 2001 |
PNA PROBES, PROBE SETS, METHODS AND KITS PERTAINING TO THE
UNIVERSAL DETECTION OF BACTERIA AND EUCARYA
Abstract
This invention is related to novel PNA probes, probe sets,
methods and kits pertaining to the universal detection of bacteria
and/or eucarya. Preferred universal probes for the detection of
bacteria comprise a probing nucleobase sequence selected from the
group consisting of CTG-CCT-CCC-GTA-GGA; TAC-CAG-GGT-ATC-TAA-T;
CAC-GAG-CTG-ACG-ACA and CCG-ACA-AGG-AAT-TTC. Preferred universal
probes for the detection of eucarya comprise a probing nucleobase
sequence selected from the group consisting of
ACC-AGA-CTT-GCC-CTC-C; GGG-CAT-CAC-AGA-CCT-G; TAG-AAA-GGG-CAG-GGA
and TAC-AAA-GGG-CAG-GGA. The PNA probes, probe sets, methods and
kits of this invention are particularly well suited for use in
multiplex PNA-FISH assays wherein the bacteria and/or eucarya in a
sample can be individually detected, identified or quantitated.
Using exemplary assays described herein, the total number of colony
forming units (CFU) of bacteria and/or eucarya can be rapidly
determined.
Inventors: |
HYLDIG-NIELSEN, JENS J.;
(HOLLISTON, MA) ; O'KEEFE, HEATHER P.; (LEXINGTON,
MA) |
Correspondence
Address: |
BOSTON PROBES, INC
75 E WIGGINS AVENUE
BEDFORD
MA
01730
|
Assignee: |
BOSTON PROBES INC.
|
Family ID: |
26790415 |
Appl. No.: |
09/368089 |
Filed: |
August 3, 1999 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60095628 |
Aug 7, 1998 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
536/23.1 |
Current CPC
Class: |
C12Q 1/6888 20130101;
C12Q 2525/107 20130101; C12Q 1/6841 20130101; C07K 14/003
20130101 |
Class at
Publication: |
435/6 ;
536/23.1 |
International
Class: |
C12Q 001/68 |
Claims
We claim:
1. A PNA probe comprising a probing nucleobase sequence suitable
for the universal yet specific detection, identification or
enumeration of bacteria or eucarya in a sample.
2. The PNA probe of claim 1, wherein at least a portion of the
probing nucleobase sequence is at least ninety percent homologous
to the nucleobase sequences, or their complements, selected from
the group consisting of: CTG-CCT-CCC-GTA-GGA;
TAC-CAG-GGT-ATC-TAA-T; CAC-GAG-CTG-ACG-ACA; CCG-ACA-AGG-AAT-TTC;
ACC-AGA-CTT-GCC-CTC-C; GGG-CAT-CAC-AGA-CCT-G; TAG-AAA-GGG-CAG-GGA
and TAC-AAA-GGG-CAG-GGA.
3. The PNA probe of claim 1, wherein the probing nucleobase
sequence is 7-16 subunits in length.
4. The PNA probe of claim 1, wherein the probe is unlabeled.
5. The PNA probe of claim 1, wherein the probe is labeled with at
least one detectable moiety.
6. The PNA probe of claim 5, wherein the detectable moiety or
moieties are selected from the group consisting of: a dextran
conjugate, a branched nucleic acid detection system, a chromophore,
a fluorophore, a spin label, a radioisotope, an enzyme, a hapten,
an acridinium ester and a chemiluminescent compound.
7. The PNA probe of claim 1, wherein the probe is labeled with at
least two independently detectable moieties.
8. The PNA probe of claim 7, wherein the two or more independently
detectable moieties are independently detectable fluorophores.
9. The PNA probe of claim 7, wherein the two or more independently
detectable moieties are orthogonal labels.
10. The PNA probe of claim 1, wherein the probe is support
bound.
11. A PNA probe set suitable for the universal yet specific
detection, identification or quantitation of bacteria or eucarya in
a sample.
12. The probe set of claim 11, wherein at least one PNA probe of
the set has a probing nucleobase sequence of which at least a
portion is at least ninety percent homologous to the nucleobase
sequences, or their complements, selected from the group consisting
of: CTG-CCT-CCC-GTA-GGA; TAC-CAG-GGT-ATC-TAA-T;
CAC-GAG-CTG-ACG-ACA; CCG-ACA-AGG-AAT-TTC; ACC-AGA-CTT-GCC-CTC-C;
GGG-CAT-CAC-AGA-CCT-G; TAG-AAA-GGG-CAG-GGA and
TAC-AAA-GGG-CAG-GGA.
13. The probe set of claim 11, wherein at least one PNA probe of
the set is suitable for the universal yet specific detection of
bacteria and has a probing nucleobase sequence of which at least a
portion is at least ninety percent homologous to the nucleobase
sequences, or their complements, selected from the group consisting
of: CTG-CCT-CCC-GTA-GGA; TAC-CAG-GGT-ATC-TAA-T; CAC-GAG-CTG-ACG-ACA
and CCG-ACA-AGG-AAT-TTC.
14. The probe set of claim 11, wherein at least one PNA probe of
the set is suitable for the universal yet specific detection of
eucarya and has a probing nucleobase sequence of which at least a
portion is at least ninety percent homologous to the nucleobase
sequences, or their complements, selected from the group consisting
of: ACC-AGA-CTT-GCC-CTC-C; GGG-CAT-CAC-AGA-CCT-G;
TAG-AAA-GGG-CAG-GGA and TAC-AAA-GGG-CAG-GGA.
15. The probe set of claim 11, wherein one of more of the PNA
probes of the set have a probing nucleobase sequence of 7-16
subunits in length.
16. The probe set of claim 11, wherein in situ hybridization is
used to detect, identify or quantitate the bacteria and/or eucarya
in the sample.
17. The probe set of claim 11, wherein the probes of the set are
unlabeled.
18. The probe set of claim 11, wherein one or more probes of the
set are labeled with one or more detectable moieties.
19. The probe set of claim 11, wherein two or more probes of the
set are independently detectable.
20. The probe set of claim 11, wherein one or more probes of the
set are labeled with two or more independently detectable
moieties.
21. The probe set of claim 20, wherein the two or more
independently detectable moieties are orthogonal labels on the same
probe.
22. The probe set of claim 21, wherein the orthogonal labels are
each independently selected from the group consisting of
fluorophores, enzymes and haptens.
23. The probe set of claim 11, wherein at least one probe of the
set is support bound.
24. A method for detecting, identifying or quantitating bacteria
and/or eucarya in a sample, said method comprising: a. contacting
the sample with one or more PNA probes having a probing nucleobase
sequence which is specific and yet universal for bacteria or
eucarya; and b. detecting, identify or quantitating hybridization
of the probing nucleobase sequence of PNA probes to the target
sequences of bacteria or eucarya, and correlating the result with
the presence, absence or number of the bacteria or eucarya in the
sample.
25. The method of claim 24, wherein at least one PNA probe has a
probing nucleobase sequence of which at least a portion is at least
ninety percent homologous to the nucleobase sequences, or their
complements, selected from the group consisting of:
CTG-CCT-CCC-GTA-GGA; TAC-CAG-GGT-ATC-TAA-T; CAC-GAG-CTG-ACG-ACA;
CCG-ACA-AGG-AAT-TTC; ACC-AGA-CTT-GCC-CTC-C; GGG-CAT-CAC-AGA-CCT-G;
TAG-AAA-GGG-CAG-GGA and TAC-AAA-GGG-CAG-GGA.
26. The method of claim 24, wherein bacteria are detected by: a.
contacting the sample with one or more PNA probes, wherein at least
one probe of the set has a probing nucleobase sequence of which at
least a portion is at least ninety percent homologous to the
nucleobase sequences, or their complements, selected from the group
consisting of: CTG-AAT-CCA-GGA-GCA; AAC-TTG-CTG-AAC-CAC;
CCA-TCG-CAT-CTA-ACA; and TCT-AGT-CAG-TCA-GTT; and b. detecting,
identify or quantitating hybridization of the probing nucleobase
sequence of PNA probes to the target sequences and correlating the
result with the presence, absence or number of bacteria in the
sample.
27. The method of claim 24, wherein eucarya are detected by: a.
contacting the sample with one or more PNA probes, wherein at least
one probe of the set has a probing nucleobase sequence of which at
least a portion is at least ninety percent homologous to the
nucleobase sequences, or their complements, selected from the group
consisting of: ACC-AGA-CTT-GCC-CTC-C; GGG-CAT-CAC-AGA-CCT-G;
TAG-AAA-GGG-CAG-GGA and TAC-AAA-GGG-CAG-GGA; and b. detecting,
identify or quantitating hybridization of the probing nucleobase
sequence of PNA probes to the target sequences and correlating the
result with the presence, absence or number of eucarya in the
sample.
28. The method of claim 24, wherein one of more of the PNA probes
of the set have a probing nucleobase sequence of 7-16 subunits in
length.
29. The method of claim 24, wherein in situ hybridization is used
to detect, identify and/or enumerate bacteria or eucarya in the
sample.
30. The method of claim 24, wherein the probes of the set are
unlabeled.
31. The method of claim 24, wherein one or more probes of the set
are labeled with one or more detectable moieties.
32. The method of claim 24, wherein two or more probes of the set
are independently detectable.
33. The method of claim 24, wherein one or more probes of the set
are labeled with two or more independently detectable moieties.
34. The method of claim 33, wherein the two or more independently
detectable moieties are orthogonal labels.
35. The method of claim 34, wherein the orthogonal labels are each
independently selected from the group consisting of fluorophores,
enzymes and haptens.
36. The method of claim 24, wherein at least one probe of the set
is support bound.
37. A kit suitable for performing an assay which detects,
identifies or enumerates bacteria or eucarya in a sample, said kit
comprising: a. one or more PNA probes, wherein the PNA probes each
having a probing nucleobase sequence of which at least a portion is
at least ninety percent homologous to the nucleobase sequences, or
their complements, selected from the group consisting of:
CTG-CCT-CCC-GTA-GGA; TAC-CAG-GGT-ATC-TAA-T; CAC-GAG-CTG-ACG-ACA;
CCG-ACA-AGG-AAT-TTC; ACC-AGA-CTT-GCC-CTC-C; GGG-CAT-CAC-AGA-CCT-G;
TAG-AAA-GGG-CAG-GGA and TAC-AAA-GGG-CAG-GGA; and b. other reagents
or compositions necessary to perform the assay.
38. The kit of claim 37, wherein one of more of the PNA probes of
the kit have a probing nucleobase sequence of 7-16 subunits in
length.
39. The kit of claim 37, wherein one or more probes of the kit are
unlabeled.
40. The kit of claim 37, wherein at least one or more probes or the
kit are labeled with one or more detectable moieties.
41. The kit of claim 40, wherein the detectable moiety or moieties
are selected from the group consisting of: a dextran conjugate, a
branched nucleic acid detection system, a chromophore, a
fluorophore, a spin label, a radioisotope, an enzyme, a hapten, an
acridinium ester and a chemiluminescent compound.
42. The kit of claim 37, wherein two or more probes of the kit are
independently detectable.
43. The kit of claim 42, wherein the independently detectable
probes of the kit are used to distinguish between bacteria and
eucarya in the same sample.
44. The kit of claim 43, wherein at least one probe is labeled with
at least two independently detectable moieties.
45. The kit of claim 44, wherein the two or more independently
detectable moieties are orthogonal labels.
46. The kit of claim 45, wherein the orthogonal labels are each
independently selected from the group consisting of fluorophores,
enzymes and haptens.
47. The kit of claim 39, wherein hybridization of the probing
nucleobase sequence of unlabeled probes to the nucleic acid of
bacteria or eucarya of interest is detected using an antibody or
antibody fragment, wherein the antibody or antibody fragment
specifically binds, under antibody binding conditions, to the
PNA/nucleic acid complex formed.
48. The kit of claim 47, comprising an antibody labeled with one or
more detectable moieties.
49. The kit of claim 48, wherein the one or more detectable
moieties are selected from the group consisting of a dextran
conjugate, a branched nucleic acid detection system, a chromophore,
a fluorophore, a spin label, a radioisotope, an enzyme, a hapten,
an acridinium ester and a chemiluminescent compound.
50. The kit of claim 37, wherein the assay is an in-situ
hybridization assay.
51. The kit of claim 37, wherein the kit is used to detect bacteria
or eucarya in food, beverages, water, pharmaceutical products,
personal care products, dairy products or environmental
samples.
52. The kit of claim 51, wherein the kit is used to test raw
materials, products or processes.
53. A multiplex PNA-ISH assay for the detection, identification or
quantitation of bacteria and eucarya.
54. The assay of claim 53, wherein at least one probe used in the
assay has a probing nucleobase sequence of which at least a portion
is at least ninety percent homologous to the nucleobase sequences,
or their complements, selected from the group consisting of:
CTG-CCT-CCC-GTA-GGA; TAC-CAG-GGT-ATC-TAA-T; CAC-GAG-CTG-ACG-ACA and
CCG-ACA-AGG-AAT-TTC; and at least one other probe has a probing
nucleobase sequence of which at least a portion is at least ninety
percent homologous to the nucleobase sequences, or their
complements, selected from the group consisting of:
ACC-AGA-CTT-GCC-CTC-C; GGG-CAT-CAC-AGA-CCT-G; TAG-AAA-GGG-CAG-GGA
and TAC-AAA-GGG-CAG-GGA.
55. The assay of claim 43, wherein independently detectable
fluorophores linked to the probes are used to distinguish between
bacteria and eucarya.
56. A method for identifying and enumerating bacteria and/or
eucarya in a liquid sample; said method comprising: a. filtering a
fixed volume of liquid using a filter having a pore size which does
not allow bacteria and eucarya of interest to freely pass; b.
incubating the filter containing the organisms under suitable
culture conditions; c. fixing the micro-colonies of organisms to
the filter; d. contacting the filter bound micro-colonies of
bacteria and/or eucarya, under suitable in-situ hybridization
conditions with appropriate specific yet universal PNA probes to
thereby form one or more PNA probe/target sequence hybrids; e.
detecting the presence of PNA probe/target sequence hybrids formed;
and f. identifying the bacteria and eucarya in the sample by
correlation of the presence of specific probe/target sequence
hybrids in the sample.
57. The method of claim 56, wherein the PNA probe/target sequence
hybrids are detected using a labeled antibody to the PNA
probe/target sequence hybrids.
58. The method of claim 56, wherein the independently detectable
labels are linked to the PNA probes so as to enable the independent
identification and enumeration of the bacteria and eucarya.
59. A PNA probe comprising two or more linked orthogonal
labels.
60. The PNA probe of claim 59, wherein the orthogonal labels are
each independently selected from the group consisting of
fluorophores, enzymes and haptens.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Serial No. 60/095,628 filed on Aug. 7, 1998.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention is related to the field of probe-based
detection, analysis and quantitation of bacteria and eucarya. More
specifically, this invention relates to novel PNA probes, probe
sets, methods and kits which can be used to detect, identify or
quantitate one or more bacteria and/or eucarya which may be present
in a sample.
[0004] 2. Description of the Related Art
[0005] Nucleic acid hybridization is a fundamental process in
molecular biology. Probe-based assays are useful in the detection,
quantitation and analysis of nucleic acids. Nucleic acid probes
have long been used to analyze samples for the presence of nucleic
acid from bacteria, fungi, virus or other organisms and are also
useful in examining genetically-based disease states or clinical
conditions of interest. Nonetheless, probe-based assays have been
slow to achieve commercial success. This lack of commercial success
is, at least partially, the result of difficulties associated with
specificity, sensitivity and reliability.
[0006] Hybridization assays hold promise as a means to screen large
numbers of samples for conditions of interest. In practice,
however, it is often difficult to multiplex a hybridization assay
given the requirement that each of the many very different probes
in the assay must exhibit a very high degree of specificity for a
specific target nucleic acid under the same or similar conditions
of stringency. Given the difficulties in specificity, sensitivity
and reliability of nucleic acid probes in assays designed to detect
a single target nucleic acid, sensitive and reliable methods for
the multiplex analysis of samples have been particularly
elusive.
[0007] The in-situ targeting of rRNA as a means to detect, identify
or quantitate organisms is well established (See: Amann et al.,
Microbiological Reviews, 59: 143-169 (1995). Nucleic acid probes
for the universal detection of bacteria and eucarya having the same
or similar nucleobase composition to the PNA probes claimed herein
can be found in Table 3 of Amann et al. The table lists probes and
nucleic acid sequences derived from relevant scientific
literature.
[0008] Despite its name, Peptide Nucleic Acid (PNA) is neither a
peptide, a nucleic acid nor is it an acid. Peptide Nucleic Acid
(PNA) is a non-naturally occurring polyamide which can hybridize to
nucleic acid (DNA and RNA) with sequence specificity (See: U.S.
Pat. No. 5,539,082 and Egholm et al., Nature 365: 566-568 (1993)).
Being a non-naturally occurring molecule, unmodified PNA is not
known to be a substrate for the enzymes which are known to degrade
peptides or nucleic acids. Therefore, PNA should be stable in
biological samples, as well as have a long shelf-life. Unlike
nucleic acid hybridization which is very dependent on ionic
strength, the hybridization of a PNA with a nucleic acid is fairly
independent of ionic strength and is favored at low ionic strength,
conditions which strongly disfavor the hybridization of nucleic
acid to nucleic acid (Egholm et al., Nature, at p. 567). The effect
of ionic strength on the stability and conformation of PNA
complexes has been extensively investigated (Tomac et al., J. Am.
Chem. Soc. 118:55 44-5552 (1996)). Sequence discrimination is more
efficient for PNA recognizing DNA than for DNA recognizing DNA
(Egholm et al., Nature, at p. 566). However, the advantages in
point mutation discrimination with PNA probes, as compared with DNA
probes, in a hybridization assay, appears to be somewhat sequence
dependent (Nielsen et al., Anti-Cancer Drug Design 8:53-65, (1993)
and Weiler et al., Nucl. Acids Res. 25: 2792-2799 (1997)).
[0009] Though they hybridize to nucleic acid with sequence
specificity (See: Egholm et al., Nature, at p. 567), PNAs have been
slow to achieve commercial success at least partially due to cost,
sequence specific properties/problems associated with solubility
and self-aggregation (See: Bergman, F., Bannwarth, W. and Tam, S.,
Tett. Lett. 36:6823-6826 (1995), Haaima, G., Lohse, A., Buchardt,
0. and Nielsen, P. E., Angew. Chem. Int. Ed. Engl. 35:1939-1942
(1996) and Lesnik, E., Hassman, F., Barbeau, J., Teng, K. and
Weiler, K., Nucleosides & Nucleotides 16:1775-1779 (1997) at p
433, col. 1, ln. 28 through col. 2, In. 3) as well as the
uncertainty pertaining to non-specific interactions which might
occur in complex systems such as a cell (See: Good, L. et al.,
Antisense & Nucleic Acid Drug Development 7:431-437 (1997)).
Consequently, their unique properties clearly demonstrate that PNA
is not the equivalent of a nucleic acid in either structure or
function. Thus, PNA probes need to be evaluated for performance and
optimization to thereby confirm whether they can be used to
specifically and reliably detect a particular nucleic acid target
sequence, particularly when the target sequence exists in a complex
sample such as a cell, tissue or organism.
[0010] PNA probes have been demonstrated as being useful for the
detection of rRNA in ISH and FISH assays (See: WO95/32305 and
WO97/18325). PNA probes have also been used in the analysis of mRNA
(e.g. Kappa Light Chain), viral nucleic acid (e.g. human
papillomavirus) and the analysis of centromeric sequences in human
chromosomes and human telomeres (See: Lansdorp et al., Human Mol.
Genetics, 5: 685-691 (1996) as well as W097/14026). Similarly, the
analysis of trinucleotide repeats in chromosomal DNA using
appropriate PNA probes has been suggested (See: WO97/14026). A PNA
probe has also been used to detect human X chromosome specific
sequences in a PNA-FISH format (See: W097/18325).
[0011] Any method, kits or compositions which could improve the
specificity, sensitivity and reliability of probe-based assays for
the detection of microorganisms in samples of interest would be a
useful advance in the state of the art particularly where the
methods were uniformly applicable to probes of all or substantially
all sequence variations. Moreover, the methods, kits or
compositions would be particularly useful if they could provide for
the rapid, reliable and sensitive multiplex analysis of samples for
the presence or absence of microorganisms and particularly bacteria
and/or eucarya. The probes and assays would be particularly useful
if they were well suited for the detecting, identifying or
quantitating only colony forming units (viable organisms) in a
sample.
SUMMARY OF THE INVENTION
[0012] This invention is directed to PNA probes, probe sets,
methods and kits useful for the universal detection, identification
and/or enumeration of bacteria and/or eucarya in a sample. The
preferred probing nucleobase sequence of the universal probes of
this invention are listed in Table 1, below. In preferred
embodiments, PNA probes are organized into a set which is designed
to detect, identify or quantitate the bacteria and/or eucarya which
may be present in the sample. In a most preferred embodiment, the
probe set is suitable for the specific detection, identification
and/or quantitation of the total bacteria and/or total eucarya
present in a sample.
[0013] Unique PNA probe constructs of this invention also include
probes comprising two or more different types of labels such as the
use of a hapten/fluorophore (e.g. fluorescein) in combination with
an enzyme (e.g. soy bean peroxidase). Such orthogonally labeled
probes can be experimental controls used in complex analysis
systems or otherwise merely be used to provide alternative
detection methodologies.
[0014] This invention is further directed to a method suitable for
detecting, identifying or quantitating one or more bacteria and/or
one or more eucarya in a sample. The method comprises contacting
the sample with one or more PNA probes, wherein suitable probes are
described herein. According to the method, bacteria and/or eucarya
are then detected, identified or quantitated. Detection,
identification and or quantitation is made possible by correlating
the hybridization, under suitable hybridization conditions, of the
probing nucleobase sequence of a PNA probe to the target sequence
of bacteria or eucarya in the sample to thereby determine the
presence, absence or number of bacteria and/or eucarya in the
sample. This correlation is made possible by direct or indirect
detection of the probe/target sequence hybrid.
[0015] In yet another embodiment, this invention is also directed
to kits suitable for performing an assay which detects, identifies
or quantitates the bacteria and/or eucarya in a sample. The kits of
this invention comprise one or more PNA probes (as described
herein) and other reagents or compositions which are selected to
perform an assay or otherwise simplify the performance of an
assay.
[0016] The PNA probes, probe sets, methods and kits of this
invention have been demonstrated to be specific and yet universal
for either bacteria or eucarya by the Examples contained herein. By
"specific and yet universal" we mean that the PNA probes of this
invention, either alone or in combination, detect target sequences
within virtually all bacteria or eucarya, as the case may be,
without any substantial cross reaction with non-target organisms.
By "virtually all bacteria or eucarya" we mean that less than 2-4
percent of the target organisms will not be detectable with these
universal probes (based on analysis of sequence information
available in Genebank 104). By "without substantial cross reaction"
we mean that less than 2-4 percent of non-target organisms will be
detected when using these probes (based on analysis of sequence
information available in Genebank 104). Moreover, the assays
described herein are rapid (the entire assay can typically be
performed in 3 hours or less with the probe hybridization requiring
only 15-60 minutes), sensitive, reliable and generally applicable
to probes of significantly different probing nucleobase sequence
length and sequence variation. They can be used in an assay to
accurately detect, identify and/or quantitate the total bacteria
and/or total eucarya in a sample.
[0017] This invention is also directed to a multiplex PNA in-situ
hybridization (PNA-ISH) assay and particularly a PNA-FISH assay. As
a demonstration of the versatility of the PNA probes, probe sets,
methods and kits of this invention, a fluorescent in-situ
hybridization assay was multiplexed without any change to the
protocol. The assay clearly provided for the detection,
classification and/or enumeration of bacteria and eucarya in the
sample (See: Example 12, herein). Thus, Applicants have
demonstrated (believed to be the first successful example) the
feasibility of a multiplex PNA-FISH assay which is suitable for, in
a single assay, positively detecting, characterizing and
quantitating the total bacteria and/or total eucarya present in a
sample.
[0018] Since probe-based analysis detects nucleic acid without
regard to the metabolic state of the organism in which the genetic
material exists, the analysis of cells in culture is preferably
used to distinguish between viable organisms and dead (non-viable)
organisms, the presence of which are generally not considered to
cause spoilage or contamination. Consequently, this invention is
further directed to a rapid culture-based detection, identification
and/or enumeration of total viable bacteria and/or total viable
eucarya in a sample of interest.
[0019] The PNA probes, probe sets, methods and kits of this
invention are particularly useful for the detection, identification
and/or enumeration of bacteria and eucarya (e.g. pathogens) in
food, beverages, water, pharmaceutical products, personal care
products, dairy products or environmental samples. The analysis of
preferred non-limiting beverages include soda, bottled water, fruit
juice, beer, wine or liquor products. Suitable PNA probes, probe
sets, methods and kits will be particularly useful for the analysis
of raw materials, equipment, products or processes used to
manufacture or store food, beverages, water, pharmaceutical
products, personal care products dairy products or environmental
samples.
[0020] Additionally, the PNA probes, probe sets, methods and kits
of this invention are particularly useful for the detection of
bacteria and eucarya (e.g. pathogens) in clinical samples and
clinical environments. Suitable PNA probes, probe sets, methods and
kits will be particularly useful for the analysis of clinical
specimens, equipment, fixtures or products used to treat humans or
animals.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIGS. 1-I through 1-V are electronic images of dot blot
assays used to examine the individual (Panels II-V) and combined
(Panel I) universal detectability of 4 different fluorescein
labeled (fluorescein used as a hapten in the experiment) PNA
oligomers directed to target sequences in the rRNA of bacteria.
[0022] FIGS. 2-I through 2-IV are electronic images of dot blot
assays used to examine the universal detectability of 4 different
soy bean peroxidase labeled PNA oligomers directed to target
sequences in the rRNA of bacteria.
[0023] FIGS. 3-I through 3-IV are electronic images of dot blot
assays used to examine the universal detectability of 4 different
fluorescein labeled (fluorescein used as a hapten in the
experiment) PNA oligomers directed to target sequences in the rRNA
of eucarya.
[0024] FIGS. 4-I and 4-II are electronic images of dot blot assays
used to compare the detection limits of a universal bacterial PNA
probe labeled with both an enzyme (soy bean peroxidase) and a
hapten (fluorescein).
[0025] FIGS. 5-I and 5-II are electronic images of dot blot assays
used to compare the specificity of PNA probes for either eucarya
(Panel I) or bacteria (Panel II).
[0026] FIGS. 6-I through 6-IV are individual or composite digital
images of the same section of a sample slide containing bacteria
and eucarya which were treated with two universal PNA probes
wherein the PNA probes are independently detectable for either
bacteria (green fluorescence) and eucarya (red fluorescence). The
images were obtained using a fluorescence microscope and
commercially available light filters fitted to the microscope and
the camera respectively. Panel 6-I is the image obtained using a
red microscope filter and red camera filter; Panel 6-II is the
image obtained using a green microscope filter and green camera
filter; Panel 6-II is the image obtained using a triple (green, red
and blue) microscope filter (wherein the camera image records on
the image obtained with the green camera filter); and Panel 6-IV is
a digitally created composite of the images presented in Panels 6-I
and 6-II.
[0027] FIGS. 7-I and 7-II are electronic images of dot blot assays
used to compare the specificity of PNA probe mixtures for either
eucarya (Panel I) or bacteria (Panel II).
[0028] FIGS. 8I-III are electronic images of X-ray film analysis of
colonies of yeast and bacteria grown from organisms isolated from a
liquid sample using a round membrane filter wherein the colonies
are detected using in-situ hybridization with the SBP-labeled PNA
probes, specific yet universal for either bacteria or eucarya,
directly on the membrane filter from which the organisms were
isolated and colonies grown.
DETAILED DESCRIPTION OF THE INVENTION
[0029] 1. Definitions:
[0030] a. As used herein, the term "nucleobase" shall include those
naturally occurring and those non-naturally occurring heterocyclic
moieties commonly known to those who utilize nucleic acid
technology or utilize peptide nucleic acid technology to thereby
generate polymers which can sequence specifically bind to nucleic
acids.
[0031] b. As used herein, the term "nucleobase sequence" is any
segment of a polymer which comprises nucleobase containing
subunits. Non-limiting examples of suitable polymers or polymers
segments include oligonucleotides, oligoribonucleotides, peptide
nucleic acids, nucleic acid analogs, nucleic acid mimics or
chimeras.
[0032] c. As used herein, the term "target sequence" is the nucleic
acid nucleobase sequence of bacteria and/or eucarya which is to be
detected in an assay and to which at least a portion of the probing
nucleobase sequence is designed to hybridize.
[0033] d. As used herein, the term "peptide nucleic acid" or "PNA"
shall be defined as any oligomer, linked polymer or chimeric
oligomer, comprising two or more PNA subunits (residues), including
any of the compounds referred to or claimed as peptide nucleic
acids in U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331,
5,736,336, 5,773,571 or 5,786,461 (all of which are herein
incorporated by reference). The term "peptide nucleic acid" or
"PNA" shall also apply to polymers comprising two or more subunits
of those nucleic acid mimics described in the following
publications: Diderichsen et al., Tett. Lett. 37: 475-478 (1996);
Fujii et al., Bioorg. Med. Chem. Lett. 7: 637-627 (1997); Jordan et
al., Bioorg. Med. Chem. Lett. 7: 687-690 (1997); Krotz et al.,
Tett. Lett. 36: 6941-6944 (1995); Lagriffoul et al., Bioorg. Med.
Chem. Lett. 4: 1081-1082 (1994); Lowe et al., J. Chem. Soc. Perkin
Trans. 1, (1997) 1: 539-546; Lowe et al., J. Chem. Soc. Perkin
Trans. 11: 547-554 (1997); Lowe et al., J. Chem. Soc. Perkin Trans.
1 1:5 55-560 (1997); Petersen et al., Bioorg. Med. Chem. Lett. 6:
793-796 (1996); Diederichsen, U., Bioorganic & Med. Chem.
Lett., 8: 165-168 (1998); Cantin et al., Tett. Lett., 38: 4211-4214
(1997); Ciapetti et al., Tetrahedron, 53: 1167-1176 (1997);
Lagriffoule et al., Chem. Eur. J., 3: 912-919 (1997) and WIPO
patent application WO96/04000 by Shah et al. and entitled
"Peptide-based nucleic acid mimics (PENAMs)".
[0034] In preferred embodiments, a PNA is a polymer comprising two
or more subunits of the formula: 1
[0035] wherein, each J is the same or different and is selected
from the group consisting of H, R.sup.1, OR.sup.1, SR.sup.1,
NHR.sup.1, NR.sup.1.sub.2, F, Cl, Br and I. Each K is the same or
different and is selected from the group consisting of O, S, NH and
NR.sup.1. Each R.sup.1 is the same or different and is an alkyl
group having one to five carbon atoms which may optionally contain
a heteroatom or a substituted or unsubstituted aryl group. Each A
is selected from the group consisting of a single bond, a group of
the formula; --(CJ.sub.2).sub.s- and a group of the formula;
--(CJ.sub.2).sub.sC(O)--, wherein, J is defined above and each s is
an whole number from one to five. The whole number t is 1 or 2 and
the whole number u is 1 or 2. Each L is the same or different and
is independently selected from the group consisting of J, adenine,
cytosine, guanine, thymine, uridine, 5-methylcytosine,
2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine,
hypoxanthine, pseudoisocytosine, 2-thiouracil, 2-thiothymidine,
other naturally occurring nucleobase analogs, other non-naturally
occurring nucleobases, substituted and unsubstituted aromatic
moieties, biotin, fluorescein and dabcyl. In the most preferred
embodiment, a PNA subunit consists of a naturally occurring or
non-naturally occurring nucleobase attached to the aza nitrogen of
the N-[2-(aminoethyl)]glycine backbone through a methylene carbonyl
linkage.
[0036] e. As used herein, the terms "label" and "detectable moiety"
shall be interchangeable and shall refer to moieties which can be
attached to a PNA probe, antibody or antibody fragment to thereby
render the probe, antibody or antibody fragment detectable by an
instrument or method.
[0037] f. As used herein, the term "chimera" or "chimeric oligomer"
shall mean an oligomer comprising two or more linked subunits which
are selected from different classes of subunits. For example, a
PNA/DNA chimera would comprise at least two PNA subunits linked to
at least one 2'-deoxyribonucleic acid subunit (For exemplary
methods and compositions related to PNA/DNA chimera preparation
See: WO96/40709). Exemplary component subunits of the chimera are
selected from the group consisting of PNA subunits, naturally
occurring amino acid subunits, DNA subunits, RNA subunits and
subunits of analogues or mimics of nucleic acids.
[0038] g. As used herein, the term "linked polymer" shall mean a
polymer comprising two or more polymer segments which are linked by
a linker. The polymer segments which are linked to form the linked
polymer are selected from the group consisting of an
oligodeoxynucleotide, an oligoribonucleotide, a peptide, a
polyamide, a peptide nucleic acid (PNA) and a chimera.
[0039] 2. Description
[0040] I. General:
[0041] PNA Synthesis:
[0042] Methods for the chemical assembly of PNAs are well known
(See: U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331,
5,736,336, 5,773,571 or 5,786,571, herein incorporated by
reference). Chemicals and instrumentation for the support bound
automated chemical assembly of peptide nucleic acids are now
commercially available. Both labeled and unlabeled PNA oligomers
are likewise available from commercial vendors of custom PNA
oligomers. Chemical assembly of a PNA is analogous to solid phase
peptide synthesis, wherein at each cycle of assembly the oligomer
possesses a reactive alkyl amino terminus which is condensed with
the next synthon to be added to the growing polymer. Because
standard peptide chemistry is utilized, natural and non-natural
amino acids are routinely incorporated into a PNA oligomer. Because
a PNA is a polyamide, it has a C-terminus (carboxyl terminus) and
an N-terminus (amino terminus). For the purposes of the design of a
hybridization probe suitable for antiparallel binding to the target
sequence (the preferred orientation), the N-terminus of the probing
nucleobase sequence of the PNA probe is the equivalent of the
5'-hydroxyl terminus of an equivalent DNA or RNA
oligonucleotide.
[0043] PNA Labeling:
[0044] Preferred non-limiting methods for labeling non-nucleic acid
probes and PNAs are described in WO98/24933, WO99/22018,
WO99/21881, WO99/37670; copending and co-owned applications U.S.
Ser. Nos. 09/179,298, 09/179,162, 09/225,048 and 09/275,848 (herein
incorporated by reference), the priority documents listed as
related applications, the examples section of this specification or
are otherwise well known in the art of PNA synthesis (See: Nielsen,
P. E., Egholm, M., Peptide Nucleic Acids, Horizon Scientific Press
(1999) pp. 81-86).
[0045] Labels:
[0046] Non-limiting examples of detectable moieties (labels)
suitable for labeling PNA probes or antibodies used in the practice
of this invention would include a dextran conjugate, a branched
nucleic acid detection system, a chromophore, a fluorophore, a spin
label, a radioisotope, an enzyme, a hapten, an acridinium ester and
a chemiluminescent compound. Other suitable labeling reagents and
preferred methods of attachment would be recognized by those of
ordinary skill in the art of PNA, peptide or nucleic acid
synthesis.
[0047] Preferred haptens include 5(6)-carboxyfluorescein,
2,4-dinitrophenyl, digoxigenin, and biotin.
[0048] Preferred fluorochromes (fluorophores) include
5(6)-carboxyfluorescein (Flu),
6-((7-amino-4-methylcoumarin-3-acetyl)amin- o)hexanoic acid (Cou),
5(and 6)-carboxy-X-rhodamine (Rox), Cyanine 2 (Cy2) Dye, Cyanine 3
(Cy3) Dye, Cyanine 3.5 (Cy3.5) Dye, Cyanine 5 (Cy5) Dye, Cyanine
5.5 (Cy5.5) Dye Cyanine 7 (Cy7) Dye, Cyanine 9 (Cy9) Dye (Cyanine
dyes 2, 3, 3.5, 5 and 5.5 are available as NHS esters from
Amersham, Arlington Heights, Ill.) or the Alexa dye series
(Molecular Probes, Eugene, Ore.).
[0049] Preferred enzymes include polymerases (e.g. Taq polymerase,
Klenow DNA polymerase, T7 DNA polymerase, Sequenase, DNA polymerase
1 and phi29 polymerase), alkaline phosphatase (AP), horseradish
peroxidase (HRP) and most preferably, soy bean peroxidase
(SBP).
[0050] Detectable and Independently Detectable Moieties/Multiplex
Analysis:
[0051] In preferred embodiments of this invention, a multiplex
hybridization assay is performed. In a multiplex assay, numerous
conditions of interest are simultaneously examined. Multiplex
analysis relies on the ability to sort sample components or the
data associated therewith, during or after the assay is completed.
In preferred embodiments of the invention, one or more distinct
independently detectable moieties are used to label two or more
different probes used in an assay. The ability to differentiate
between and/or quantitate each of the independently detectable
moieties provides the means to multiplex a hybridization assay
because the data which correlates with the hybridization of each of
the distinctly (independently) labeled probe to a particular target
sequence can be correlated with the presence, absence or quantity
of each organism sought to be detected in the sample. Consequently,
the multiplex assays of this invention may be used to
simultaneously detect the presence, absence or quantity of two or
more organisms in the same sample and in the same assay.
[0052] Spacer/Linker moieties:
[0053] Generally, spacers are used to minimize the adverse effects
that bulky labeling reagents might have on hybridization properties
of probes. Linkers typically induce flexibility and randomness into
the probe or otherwise link two or more nucleobase sequences of a
probe or component polymer. Preferred spacer/linker moieties for
the nucleobase polymers of this invention consist of one or more
aminoalkyl carboxylic acids (e.g. aminocaproic acid) the side chain
of an amino acid (e.g. the side chain of lysine or ornithine)
natural amino acids (e.g. glycine), aminooxyalkylacids (e.g.
8-amino-3,6-dioxaoctanoic acid), alkyl diacids (e.g. succinic
acid), alkyloxy diacids (e.g. diglycolic acid) or alkyldiamines
(e.g. 1,8-diamino-3,6-dioxaoctane). Spacer/linker moieties may also
incidentally or intentionally be constructed to improve the water
solubility of the probe (For example see: Gildea et al., Tett.
Lett. 39: 7255-7258 (1998)). Preferably, a spacer/linker moiety
comprises one or more linked compounds having the formula:
-Y-(O.sub.m-(CW.sub.2).s- ub.n).sub.o-Z-. The group Y is selected
from the group consisting of: a single bond, -(CW.sub.2).sub.p-,
--C(O)(CW.sub.2).sub.p-, --C(S)(CW.sub.2).sub.p- and
--S(O.sub.2)(CW.sub.2).sub.p. The group Z has the formula NH,
NR.sup.2, S or O. Each W is independently H, R.sup.2, --OR.sup.2,
F, Cl, Br or I; wherein, each R.sup.2 is independently selected
from the group consisting of: --CX.sub.3, --CX.sub.2CX.sub.3,
--CX.sub.2CX.sub.2CX.sub.3, --CX.sub.2CX(CX.sub.3).sub.2, and
--C(CX.sub.3).sub.3. Each X is independently H, F, Cl, Br or I.
Each m is independently 0 or 1. Each n, o and p are independently
integers from 0 to 10.
[0054] Hybridization Conditions/Stringency:
[0055] Those of ordinary skill in the art of nucleic acid
hybridization will recognize that factors commonly used to impose
or control stringency of hybridization include formamide
concentration (or other chemical denaturant reagent), salt
concentration (i.e., ionic strength), hybridization temperature,
detergent concentration, pH and the presence or absence of
chaotropes. Optimal stringency for a probe/target combination is
often found by the well known technique of fixing several of the
aforementioned stringency factors and then determining the effect
of varying a single stringency factor. The same stringency factors
can be modulated to thereby control the stringency of hybridization
of a PNA to a nucleic acid, except that the hybridization of a PNA
is fairly independent of ionic strength. Optimal stringency for an
assay may be experimentally determined by examination of each
stringency factor until the desired degree of discrimination is
achieved.
[0056] Suitable Hybridization Conditions:
[0057] Generally, the more closely related the background causing
nucleic acid contaminates are to the target sequence, the more
carefully stringency must be controlled. Blocking probes may also
be used as a means to improve discrimination beyond the limits
possible by mere optimization of stringency factors. Suitable
hybridization conditions will thus comprise conditions under which
the desired degree of discrimination is achieved such that an assay
generates an accurate (within the tolerance desired for the assay)
and reproducible result. Aided by no more than routine
experimentation and the disclosure provided herein, those of skill
in the art will easily be able to determine suitable hybridization
conditions for performing assays utilizing the methods and
compositions described herein. Suitable in-situ hybridization
conditions are those conditions suitable for performing an in-situ
hybridization procedure. Thus, suitable in-situ hybridization
conditions will become apparent using the disclosure and references
herein; with or without additional routine experimentation.
[0058] Blocking Probes:
[0059] Blocking probes are nucleic acid or non-nucleic acid probes
which can be used to suppress the binding of the probing nucleobase
sequence of the probing polymer to a non-target sequence. Preferred
blocking probes are PNA probes (See: Coull et al., WIPO publication
No. WO98/24933). Typically blocking probes are closely related to
the probing nucleobase sequence and preferably they comprise a
point mutation of the probing segment. It is believed that blocking
probes operate by hybridization to the non-target sequence to
thereby form a more thermodynamically stable complex than is formed
by hybridization between the probing nucleobase sequence and the
non-target sequence. Formation of the more stable and preferred
complex blocks formation of the less stable non-preferred complex
between the probing nucleobase sequence and the non-target
sequence. Thus, blocking probes can be used with the methods, kits
and compositions of this invention to suppress the binding of the
nucleic acid or non-nucleic acid probe to a non-target sequence
which might be present and interfere with the performance of the
assay. Blocking probes are particularly advantageous in single
point mutation discrimination.
[0060] Probing Nucleobase Sequence:
[0061] The probing nucleobase sequence of a PNA probe is the
specific sequence recognition portion of the construct. Therefore,
the probing nucleobase sequence is a sequence of PNA subunits
designed to hybridize to a target sequence to thereby be used to
detect the presence, absence or number of organisms of interest in
a sample. Consequently, with due consideration of the requirements
of a PNA probe for the assay format chosen and the organism sought
to be detected, the length of the probing nucleobase sequence of
the PNA probe will generally be chosen such that a stable complex
is formed with the target sequence under suitable hybridization
conditions or suitable in-situ hybridization conditions.
[0062] The probing nucleobase sequence suitable for detecting the
target organism listed in the table, will generally, but not
necessarily, have a length of 16 or fewer PNA subunits (most
preferably 7-16 subunits in length) wherein at least a portion of
the probing nucleobase sequence is at least ninety percent
homologous to the nucleobase sequences listed in Table 1, or their
complements. Longer probing nucleobase sequences may be used but
they are not preferred. Complements of the probing nucleobase
sequence are included since it is possible to prepare or amplify
copies of the target sequence wherein the copies are complements of
the target sequence and thus, will bind to the complement of the
probing nucleobase sequences listed in Table 1. The most preferred
probing nucleobase sequences are listed in Table 1. These probing
nucleobase sequences have been shown to be specific yet universal
for the target organism identified in the table.
[0063] The probing nucleobase sequence of a PNA probe will
generally be complementary to the target sequence. Alternatively, a
substantially complementary probing nucleobase sequence might be
used since it has been demonstrated that greater sequence
discrimination can be obtained when utilizing probes wherein there
exists one or more point mutations (base mismatch) between the
probe and the target sequence (See: Guo et al., Nature
Biotechnology 15:331-335 (1997)). For example, probing nucleobase
sequences 7 and 8 of Table 1 differ by a single nucleobase (e.g. a
point mutation). It is also noteworthy that probing nucleobase
sequences 7 and 8 are particularly purine rich and the literature
teaches that such PNA sequences are difficult or impossible to
purify and characterize (See: Nielsen, P. E., Egholm, M., Peptide
Nucleic Acids, Horizon Scientific Press (1999) p 253). Table 3 of
Amann et al. identifies a nucleic acid probe having a nucleobase
sequence identical to the probing nucleobase sequence listed as
Sequence ID No. 7 (See: Amann et al., Microbiological Reviews, 59:
143-169 (1995). Examination of Genbank 104 indicates that Sequence
ID No. 8 is perfectly homologous to the rRNA of substantially all
eucarya for which information exists in the databank. The sequences
are related as point mutations having a 93.3 percent sequence
homology. The Examples and Figures of this specification indicate
that the both probing nucleobase sequences 7 and 8 of Table 1 are
suitable for the specific yet universal detection of eucarya. Thus,
the data presented herein supports the premise that PNA probes
having a sequence homology of ninety percent or greater are useful
equivalents of the exact probing nucleobase sequences identified in
Table 1.
[0064] This invention contemplates that variations in the probing
nucleobase sequences listed in Table 1 shall provide PNA probes
which are suitable for the specific detection of the organisms
listed. Common variations include, deletions, insertions and frame
shifts. Variation of the probing nucleobase sequences within the
parameters described herein are considered to be an embodiment of
this invention.
[0065] Probe Complexes:
[0066] In still another embodiment, two probes are designed to
hybridize to the target sequence sought to be detected to thereby
generate a detectable signal whereby the probing nucleobase
sequence of each probe comprises the complement to half or
approximately half of the complete target sequence of the bacteria
or eucarya sought to be detected in the assay. As a non-limiting
example, the probing nucleobase sequences of the two probes might
be designed using the assay as described in European Patent
Application 849,363, entitled "Method of identifying a nucleic acid
using triple helix formation of adjacently annealed probes" by H.
Orum et al. (See: EPA 849,363). Using this methodology, the probes
which hybridize to the target sequence may or may not be labeled.
However, it is the probe complex formed by the annealing of the
adjacent probes which is detected. Similar compositions comprised
solely of PNA probes have been described in copending and commonly
owned application U.S. Ser. No. 09/302,238, herein incorporated by
reference.
[0067] II. Preferred Embodiments of the Invention:
[0068] a. PNA Probes:
[0069] In one embodiment, this invention is directed to PNA probes.
The PNA probes of this invention are, alone or in combination,
suitable for detecting, identifying or quantitating bacteria and/or
eucarya in a sample. General characteristics (e.g. length, labels,
nucleobase sequences, linkers etc.) of PNA probes suitable for the
detection, identification or quantitation of bacteria and/or
eucarya have been previously described herein. The preferred
probing nucleobase sequences of PNA probes of this invention are
listed in Table 1.
1TABLE 1 Se- Target quence Probe Organ- rRNA ID No. Type ism Target
Probing Nucleobase Sequence 1 Universal Bacteria 16S
CTG-CCT-CCC-GTA-GGA 2 Universal Bacteria 16S TAC-CAG-GGT-ATC-TAA-T
3 Universal Bacteria 16S CAC-GAG-CTG-ACG-ACA 4 Universal Bacteria
23S CCG-ACA-AGG-AAT-TTC 5 Universal Eucarya 18S
ACC-AGA-CTT-GCC-CTC-C 6 Universal Eucarya 18S GGG-CAT-CAC-AGA-CCT-G
7 Universal Eucarya 18S TAG-AAA-GGG-CAG-GGA 8 Universal Eucarya 18S
TAC-AAA-GGG-CAG-GGA Note Apart from the functional Examples
described herein which have been performed to screen potential
probe sequences and thereby confirm their practical specificity in
an assay, the complementary target sequence to which the probing
nucleobase sequences in Table 1 hybridize were examined using
sequence alignment analysis of information currently available in
Genbank.
[0070] The PNA probes of this invention may comprise only a probing
nucleobase sequence (as previously described herein) or may
comprise additional moieties. Non-limiting examples of additional
moieties include detectable moieties (labels), linkers, spacers,
natural or non-natural amino acids, or other subunits of PNA, DNA
or RNA. Additional moieties may be functional or non-functional in
an assay. Generally however, additional moieties will be selected
to be functional within the design of the assay in which the PNA
probe is to be used. The preferred PNA probes of this invention are
labeled with one or more detectable moieties. In a more preferred
embodiment, one or more probes are labeled with a set of two or
more independently detectable moieties. Preferred sets of
independently detectable moieties are comprised of independently
detectable labels each individually selected from the group
consisting of fluorophores, enzymes and haptens.
[0071] Unique PNA probe constructs of this invention also include
probes comprising two or more different types of labels (orthogonal
labels) such as the use of a hapten/fluorophore (e.g. fluorescein)
in combination with an enzyme (e.g. soy bean peroxidase). Such
orthogonally labeled probes can be experimental controls used to
analyze complex analysis systems or otherwise merely be used to
provide alternative detection methodologies. By orthogonally
labeled probes we mean that the two labels are of such different
character that a completely different type of detection system can
be used to detect the orthogonal labels. For example, two
independently detectable fluorophores are not necessarily
orthogonal labels since fluorescence detection is common to the
detection system. However, fluorescence used in combination with an
enzyme is orthogonal since detection of the two labels can require
completely different detection methodologies.
[0072] It is an important feature of this invention that PNA probes
of this invention can be used to target both bacteria and eucarya
in the same assay under the same set of conditions. This is
surprising since the cell walls of yeast are substantially
different as compared with bacteria. Consequently, typical
conditions for in-situ analysis of yeast and bacteria using nucleic
acid probes are often substantially different. In a most preferred
embodiment of this invention, independently detectable moieties are
used to label each of at least two different PNA probes whereby at
least one probe is a universal probe for detecting bacteria and at
least one other probe is a universal probe for detecting eucarya
such that the independently detectable moieties can be used to
independently detect, identify or quantitate the bacteria and/or
the eucarya in the same sample and in the same assay. Examples 12
and 13 of this specification demonstrates the feasibility of
multiplex PNA-FISH and PNA-ISH, respectively, for the simultaneous
analysis of yeasts and bacteria present in the same sample.
[0073] In preferred embodiments, the probes of this invention are
used in in-situ hybridization (ISH) and fluorescence in-situ
hybridization (FISH) assays. Excess probe used in a ISH or FISH
assay typically must be removed so that the detectable moiety of
specifically bound probes can be detected above the background
signal which results from still present but unhybridized probe.
Generally, the excess probe is washed away after the sample has
been incubated with probe for a period of time. However, use of
dark probes are a preferred embodiment of this invention, since
there is no requirement that excess dark probe be completely
removed (washed away) from the sample since it generates little or
no detectable background.
[0074] As used herein, a "dark probe" shall be a PNA probe which
hybridizes to a nucleic acid target to thereby cause a detectable
change in at least one physical property of at least one attached
label in a manner which can be used to detect, identify or
quantitate the presence of an organism of interest in a sample of
interest. Non-limiting examples of dark probes include PNA
Molecular Beacons (See: WO99/21881, U.S. Ser. No. 08/958,532
(abandoned) and copending and commonly owned U.S. Ser. No.
09/179,298, both incorporated herein by reference) as well as
Linear Beacons (See: WO99/22018 and copending and commonly owned
U.S. Ser. No. 09/179,162, herein incorporated by reference). Thus,
changes in signal in the assay utilizing a "dark probe" can be
directly correlated with hybridization of the probing nucleobase
sequence to the target sequence of bacteria or eucarya of
interest.
[0075] Unlabeled Non-Nucleic Acid Probes:
[0076] The probes of this invention need not be labeled with a
detectable moiety to be operable within this invention. When using
the probes of this invention it is possible to detect the
probe/target sequence complex formed by hybridization of the
probing nucleobase sequence of the probe to the target sequence.
For example, a PNA/nucleic acid complex formed by the hybridization
of a PNA probing nucleobase sequence to the target sequence could
be detected using an antibody which specifically interacts with the
complex under antibody binding conditions. Suitable antibodies to
PNA/nucleic acid complexes as well as methods for their preparation
and use are described in WIPO Patent Application WO95/17430 and
U.S. Pat. No. 5,612,458, herein incorporated by reference.
[0077] The antibody/PNA/nucleic acid complex formed by interaction
of the .alpha.-PNA/nucleic acid antibody with the PNA/nucleic acid
complex can be detected by several methods. For example, the
.alpha.-PNA/nucleic acid antibody could be labeled with a
detectable moiety. Suitable detectable moieties have been
previously described herein. Thus, the presence, absence or
quantity of the detectable moiety is correlated with the presence,
absence or quantity of the antibody/PNA/nucleic acid complex and
the organism to be identified by the probing nucleobase sequence of
the PNA probe. Alternatively, the antibody/PNA/nucleic acid complex
is detected using a secondary antibody which is labeled with a
detectable moiety. Typically the secondary antibody specifically
binds to the .alpha.-PNA/nucleic acid antibody under antibody
binding conditions. Thus, the presence, absence or quantity of the
detectable moiety is correlated with the presence, absence or
quantity of the antibody/antibody/PNA/nucleic acid complex and the
organism to be identified by the probing nucleobase sequence of the
probe. As used herein, the term antibody shall include antibody
fragments which specifically bind to other antibodies or other
antibody fragments.
[0078] Immobilization of Probes To A Surface:
[0079] One or more of the PNA probes of this invention may
optionally be immobilized to a surface for the detection of the
target sequence of bacteria and/or eucarya. Generally, surface
immobilized PNA probes can be used in a capture assay. PNA probes
can be immobilized to the surface using the well known process of
UV-crosslinking. More preferably, the PNA probe is synthesized on
the surface in a manner suitable for deprotection but not cleavage
from the synthesis support (See: Weiler, J. et al, Hybridization
based DNA screening on peptide nucleic acid (PNA) oligomer arrays.,
Nucl. Acids Res., 25:2792-2799 (July, 1997)). In still another
embodiment, one or more PNA probes are covalently linked to a
surface by the reaction of a suitable functional group on the probe
with a functional group of the surface (See: Lester, A. et al, "PNA
Array Technology": Presented at Biochip Technologies Conference in
Annapolis (October, 1997)). This method is most preferred since the
PNA probes on the surface will typically be highly purified and
attached using a defined chemistry, thereby minimizing or
eliminating non-specific interactions.
[0080] Methods for the chemical attachment of probes to surfaces
generally involve the reaction of a nucleophilic group, (e.g. an
amine or thiol) of the probe to be immobilized, with an
electrophilic group on the support to be modified. Alternatively,
the nucleophile can be present on the support and the electrophile
(e.g. activated carboxylic acid) present on the probe. Because
native PNA possesses an amino terminus, a PNA will not necessarily
require modification to thereby immobilize it to a surface (See:
Lester et al., Poster entitled "PNA Array Technology").
[0081] Conditions suitable for the immobilization of a PNA probe to
a surface will generally be similar to those conditions suitable
for the labeling of the polymer. The immobilization reaction is
essentially the equivalent of labeling whereby the label is
substituted with the surface to which the polymer is to be
linked.
[0082] Numerous types of surfaces derivatized with amino groups,
carboxylic acid groups, isocyantes, isothiocyanates and malimide
groups are commercially available. Non-limiting examples of
suitable surfaces include membranes, glass, controlled pore glass,
polystyrene particles (beads), silica and gold nanoparticles.
[0083] Arrays of PNA Probes or Probe Sets:
[0084] Arrays are surfaces to which two or more probes have been
immobilized each at a specified position. Typically, the probing
nucleobase sequence of the immobilized probes is judiciously chosen
to interrogate (often using a capture assay) a sample which may
contain bacteria and/or eucarya. Because the location and
composition of each immobilized probe is known, arrays are
generally useful for the simultaneously detection, identification
or quantitation of two or more organisms which may be present in
the sample. Moreover, arrays of PNA probes may be regenerated by
stripping the hybridized nucleic acid after each assay, thereby
providing a means to repetitively analyze numerous samples using
the same array. Thus, arrays of PNA probes or PNA probe sets may be
useful for repetitive screening of samples for bacteria and/or
eucarya. The arrays of this invention comprise at least one PNA
probe (as described herein) suitable for the detection,
identification or quantitation of bacteria and/or eucarya.
Preferred probing nucleobase sequences for the immobilized PNA
probes are listed in Table 1.
[0085] Advantages of using PNA probes
[0086] It has been demonstrated that nucleic acid probes hybridize
to target sequences of rRNA (e.g. targets like 16S or 23S rRNA)
with an efficiency which is highly dependent upon whether the site
of hybridization is placed inside or outside of a highly structured
region (See: Fuchs et al., Applied and Environmental Microbiology,
64: 4973-4982 (1998)). Moving the probe just a few bases in or out
of a structured region significantly alters the overall signal
intensity. The lack of signal intensity achieved when attempting to
target structured rRNA is believed to result from the lack of probe
accessibility to the hybridization site under suitable
hybridization conditions or suitable in-situ hybridization
conditions. Furthermore, conditions which would destabilize the
structured region would simultaneously destabilize the target
sequence/probe hybrid.
[0087] When designing nucleic acid probes directed to a target
sequence which are to be used to select a target organism,
nucleobase sequence selection is further limited because rRNA is
relatively well conserved between related species. Moreover, the
limited number of sequence variations are often concentrated in the
highly structured regions of the rRNA. Therefore, some of the most
useful regions of diverse nucleobase sequence suitable for
designing organism specific probes are often unavailable to nucleic
acid probes.
[0088] Because of its unique structure, PNA probes can be designed
to target regions of rRNA under conditions of low ionic strength
wherein the secondary structure is destabilized. Because PNA probe
can efficiently, and preferably, hybridize to nucleic acid under
these conditions of low salt, the PNA probes can be designed to
target rRNA which cannot be targeted by traditional nucleic acid
probes (See: Stefano et al., Diagnostic Applications of PNA
Oligomers, Diagnostic Gene Detection and Quantification
Technologies for Infectious Agents and Human Genetic Diseases, #
948, IBC Library Series, 19-37 (1997)). Thus, the PNA probes of
this invention typically generate stronger signals than can be
achieved with nucleic acid probes of comparable nucleobase
sequence. Consequently, the PNA probes of this invention do not
suffer from the limitations characteristic of nucleic acid
probes.
[0089] b. PNA Probe Sets:
[0090] In another embodiment, this invention is directed to a PNA
probe set suitable for detecting, identifying or quantitating
bacteria and/or eucarya in a sample. The general and preferred
characteristics of PNA probes suitable for the detection,
identification or quantitation of bacteria and/or eucarya have been
previously described herein. Preferred probing nucleobase sequences
are listed in Table 1. The grouping of PNA probes within sets
characterized for specific detection, identification and/or
quantitation of either bacteria or eucarya is contemplated as a
preferred embodiment of this invention. The grouping of PNA probes
within sets characterized for specific identification and
enumeration of both bacteria and eucarya is contemplated as still
another preferred embodiment of this invention.
[0091] Probe sets of this invention shall comprise at least one PNA
probe but need not comprise only PNA probes. For example, probe
sets of this invention may comprise mixtures of PNA probes and
nucleic acid probes, provided however that a set comprises at least
one PNA probe as described herein. In preferred embodiments, some
of the probes of the set are blocking probes composed of PNA or
nucleic acid.
[0092] Table 1 lists four probing nucleobase sequences suitable for
the universal detection of bacteria and three probing nucleobase
sequences suitable for the universal detection of eucarya. Since
alternative probing nucleobase sequences exist for the detection of
either bacteria or eucarya, it is preferable to use a probe set
containing two or more PNA probes for the detection of either
bacteria or eucarya to thereby increase the detectable signal in
the assay (For Example: compare Panel I of FIG. 1 with Panels II
through V).
[0093] One exemplary probe set might therefore comprise probes
suitable for the detection of both bacteria and eucarya.
Consequently, a suitable probe set might contain at least one PNA
probe suitable for detecting bacteria and having a probing
nucleobase sequence wherein at least a portion of the probing
nucleobase sequence is at least ninety percent homologous to the
nucleobase sequence, or their complements, selected from the group
consisting of: CTG-CCT-CCC-GTA-GGA; TAC-CAG-GGT-ATC-TAA-T;
CAC-GAG-CTG-ACG-ACA and CCG-ACA-AGG-AAT-TTC; and at least one other
PNA probe suitable for detecting eucarya and having a probing
nucleobase sequence wherein at least a portion of the probing
nucleobase sequence is at least ninety percent homologous to the
nucleobase sequence, or their complements, selected from the group
consisting of: ACC-AGA-CTT-GCC-CTC-C; GGG-CAT-CAC-AGA-CCT-G;
TAG-AAA-GGG-CAG-GGA and TAC-AAA-GGG-CAG-GGA.
[0094] In a preferred embodiment, probes for detecting bacteria
would be independently detectable from probes for detecting eucarya
thereby enabling the independent or multiplex detection,
identification and/or quantitation of bacteria and eucarya in the
same sample and in the same assay. In other embodiments, the probes
are not independently detectable yet the bacteria and eucarya are
distinguishable based upon unique characteristics such as growth
rate or morphology (See: Example 13).
[0095] A second exemplary probe set might comprise only those
probes suitable for the detection of bacteria. Consequently, a
suitable probe set might contain PNA probes comprising at least
two, but preferably all, of the probing nucleobase sequences
wherein at least a portion of the probing nucleobase sequence is at
least ninety percent homologous to the nucleobase sequence, or
their complements, selected from the group consisting of:
CTG-CCT-CCC-GTA-GGA; TAC-CAG-GGT-ATC-TAA-T; CAC-GAG-CTG-ACG-ACA and
CCG-ACA-AGG-AAT-TTC.
[0096] Still a third exemplary probe set might comprise only those
probes suitable for the detection of eucarya. Consequently a
suitable probe set might contain PNA probes comprising at least
two, but preferably both, of the probing nucleobase sequences
wherein at least a portion of the probing nucleobase sequence is at
least ninety percent homologous to the nucleobase sequence, or
their complements, selected from the group consisting of:
ACC-AGA-CTT-GCC-CTC-C; GGG-CAT-CAC-AGA-CCT-G; TAG-AAA-GGG-CAG-GGA
and TAC-AAA-GGG-CAG-GGA.
[0097] Exemplary Multiplex PNA-FISH Assays:
[0098] Because the individual PNA probes of this invention can each
be labeled with independently detectable moieties, it is possible
to design PNA probe sets wherein each probe of the set is
independently detectable. The grouping of PNA probes within probe
sets characterized for detecting either or both bacteria and
eucarya is contemplated as a preferred embodiment of this
invention. For example, fluorophores which have sufficiently
different excitation and emission spectra are often used as
independently detectable moieties. Exemplary independently
detectable fluorophores are derivatives of coumarin, fluorescein
and rhodamine. Thus, an assay wherein a PNA probe set comprising
two or more PNA probes, each labeled with one of an independently
detectable moiety, could be used to independently detect, identify
or quantitate, the bacteria and eucarya in the same sample and in
the same assay. Consequently, the PNA probes, PNA probe sets,
methods and kits of this invention are particularly useful for the
rapid, sensitive, reliable and versatile multiplex analysis of
bacteria and eucarya in a single sample and/or assay. By versatile
we mean that the method is generally applicable despite substantial
variability in the nucleobase sequences of the probes or probe
length used in the assay.
[0099] Example 12 of this specification demonstrates the
feasibility of multiplex fluorescent in-situ hybridization using
independently detectable PNA probes wherein the individual bacteria
and eucarya in the sample are detectable and quantifiable. Example
13 of this specification demonstrates the feasibility of multiplex
in-situ analysis of bacteria and eucarya based upon differential
growth rates of the organisms which results in clearly
distinguishable colony morphology. Importantly, both Examples yield
information on identity and enumeration of the organisms present in
the sample using the same assay.
[0100] c. Methods:
[0101] In another embodiment, this invention is directed to a
method suitable for detecting, identifying and/or quantitating
bacteria and/or eucarya in a sample. The general and specific
characteristics of PNA probes suitable for the detection,
identification or quantitation of bacteria and/or eucarya have been
previously described herein. Preferred probing nucleobase sequences
are listed in Table 1.
[0102] The method for detecting, identifying and/or enumerating
bacteria and/or eucarya in a sample comprises contacting the sample
with one or more PNA probes suitable for hybridization to a target
sequence which is specific yet universal to bacteria or eucarya. In
preferred embodiments, the probe comprises a probing nucleobase
sequence wherein at least a portion of the probing nucleobase
sequence is at least ninety percent homologous to the nucleobase
sequence, or their complements, selected from the group consisting
of: CTG-CCT-CCC-GTA-GGA; TAC-CAG-GGT-ATC-TAA-T;
CAC-GAG-CTG-ACG-ACA; CCG-ACA-AGG-AAT-TTC; ACC-AGA-CTT-GCC-CTC-C;
GGG-CAT-CAC-AGA-CCT-G; TAG-AAA-GGG-CAG-GGA and
TAC-AAA-GGG-CAG-GGA.
[0103] According to the method, bacteria and/or eucarya in the
sample are then detected, identified and/or quantitated. Detection,
identification and/or enumeration of bacteria and/or eucarya is
made possible by correlating hybridization, under suitable
hybridization conditions or suitable in-situ hybridization
conditions, of the probing nucleobase sequence of a PNA probe to
the target sequence of the target organism sought to be detected
with the presence, absence or number of the bacteria and/or eucarya
in the sample. Typically, this correlation is made possible by
direct or indirect detection of the probe/target sequence
hybrid.
[0104] Media Based Analysis Of Bacteria and Eucarya
[0105] The methods, kits and compositions of this invention are
particularly useful for the rapid probe-based detection,
identification and/or enumeration of viable bacteria and/or
eucarya. For example, it is possible to use enzyme-linked PNA
probes in combination with in-situ analysis of colonies of bacteria
or eucarya grown directly on the medium on which they were isolated
from the sample (i.e. a filtration membrane) to thereby achieve
rapid, sensitive and specific analysis in a manner which was not
previously possible (See: Example 13).
[0106] The rapid probe-based analysis of growing bacteria and/or
eucarya requires very high sensitivity in addition to probe
specificity because the cell count is limited during the early
stages of cell division. Since this probe-based analysis detects
nucleic acid (e.g. rRNA) without regard to the metabolic state of
the organism, the analysis of growing bacteria is used to
distinguish between viable organisms and dead (non-viable)
organisms, the presence of which are not typically considered to
cause food or beverage spoilage or contamination.
[0107] Enzyme-linked probes are preferred for such analysis since
the enzymes can rapidly and repetitively turn over a substrate to
thereby achieve signal amplification suitable for high sensitivity
detection. Preferred, non-limiting, substrates include
chemiluminescent compounds, fluorophores and chromophores. PNA
probes are the preferred probe type since they hybridize rapidly to
nucleic acid and are generally more specific than nucleic acid
probes. Furthermore, PNA probes operate under conditions of low
ionic strength (favored conditions for hybridizing to structured
rRNA) and they form very stable hybrids. In-situ analysis is
preferred since viability of colony forming units (CFU) can be
absolutely determined and optionally quantitated by scoring the
colonies observed. Dead (non-viable) organisms are not scored
because they do not grow into a detectable colony.
[0108] In preferred embodiments, the bacteria are grown directly on
an isolation medium. Integration of the isolation medium with the
growth of the bacteria and/or eucarya eliminates the need for a
pre- and post- growth transfer and thereby eliminates the
opportunity for error associated therewith. Preferably, the
isolation medium is a filter or a membrane filter. Preferred
filters are microporous membrane filters such as those sold by
Millipore Corporation for the filtration of liquids. Pore sizes of
the filter are generally chosen so that the organism will not pass
though the pores thereby insuring that all the bacteria or eucarya
in the sample is collected on the filter.
[0109] After the bacteria or eucarya are grown, typically they are
fixed. Cell fixation is a term well known in the art of in-situ
hybridization and is generally, but not required as part of the
in-situ hybridization process.
[0110] Using probe-based in-situ analysis of the isolation medium,
the number of colony forming units (CFU) of bacteria and/or eucarya
which are detected by the organism specific probe, can be counted
or scored (manually or by automated methods) after an appropriate
incubation (growth) period. If the bacteria and/or eucarya grow
rapidly and the enzyme-linked PNA probes are suitable for high
sensitivity analysis, typically, the assay can be performed in 1-4
hours. If the bacteria and/or eucarya grow slowly or a less
sensitive detection method is used, the assay may require 1-7 days
depending on the rate of organism growth or differential growth
rate of the organisms of interest in the assay. Because the
bacteria and eucarya are preferably grown directly on the isolation
medium, the colonies detected are each representative of a colony
forming unit (CFU) isolated from the sample. Since the volume of
sample filtered to isolate the organisms is known and since only
viable organisms grow, the CFU's per unit volume of sample can be
directly determined.
[0111] Example 13 of this specification demonstrates using this
method for the identification and enumeration of bacteria and
eucarya in the same sample and in the same assay. With reference to
FIG. 8, soy bean peroxidase labeled PNA probes were used to detect
grown bacteria and eucarya isolated from a sample by filtration.
Because of the differential growth rate, the micro-colonies of
eucarya appear as small dots on the filtration membrane whereas the
faster growing bacteria produce very large dots (colonies) in the
same time period. Thus, multiplex analysis of the sample is
possible, even with probes having identical labeled, if the
specificity of the probes is considered in relation to other
characteristics such as the relative growth rate or colony
morphology of organisms sought to be detected, identified or
quantitated in the assay.
[0112] Exemplary Assay Formats:
[0113] The probes, probe sets, methods and kits of this invention
are suitable for the detection, identification and/or enumeration
of bacteria and/or eucarya. In preferred embodiments, in-situ
hybridization is used as the assay format for detecting identifying
or quantitating target organisms. Most preferably, in-situ
hybridization (FISH or PNA-FISH) is the assay format. Exemplary
methods for performing PNA-FISH can be found in: Thisted et al.
Cell Vision, 3:358-363 (1996) or WIPO Patent Application
WO97/18325, herein incorporated by reference. Methods used to
experimentally test specific PNA probes in PNA-FISH assays can be
found in Example 12 and 13 of this specification.
[0114] Organisms which have been treated with the probes, probe
sets or probes contained in the kits of this invention can be
detected by several exemplary methods. The cells can be fixed on
slides and then visualized with a microscope (See: Example 12),
film(See: Example 13), camera and film, luminometer or laser
scanning device. Alternatively, the cells can be fixed and then
analyzed in a flow cytometer (See for example: Lansdorp et al.;
WIPO Patent Application; WO97/14026). Automated slide scanners and
flow cytometers are particularly useful for rapidly quantitating
the number of target organisms present in a sample of interest.
[0115] d. Kits:
[0116] In yet another embodiment, this invention is directed to
kits suitable for performing an assay which detects, identifies or
quantitates bacteria and/or eucarya in a sample. The general and
preferred characteristics of PNA probes suitable for the detection,
identification or quantitation of bacteria and/or eucarya have been
previously described herein. Preferred probing nucleobase sequences
are listed in Table 1. Furthermore, methods suitable for using the
PNA probes or PNA probes sets of a kit to detect, identify or
quantitate bacteria and/or eucarya in a sample have been previously
described herein.
[0117] The kits of this invention comprise one or more PNA probes
and other reagents or compositions which are selected to perform an
assay or otherwise simplify the performance of an assay used to
detect, identify or quantitate bacteria and/or eucarya in a sample.
In kits which contain sets of PNA probes, wherein each of at least
two probes of the set are used to distinguish between and/or
enumerate the bacteria or eucarya in a sample in the same assay,
the probes of the set are preferably labeled with independently
detectable moieties so that individual bacteria or eucarya can be
detected, identified and/or enumerated. In a preferred embodiment,
PNA probes of a kit which are used to detect each of the bacteria
or eucarya are each labeled with independently detectable
fluorophores to thereby enable correlation of the presence of
signal from a particular fluorophore with the presence of one of
either the bacteria or eucarya in the sample.
[0118] e. Exemplary Applications For Using The Invention:
[0119] The PNA probes, probe sets, methods and kits of this
invention are particularly useful for the detection, identification
and/or quantitation of bacteria and eucarya (e.g. pathogens) in
food, beverages, water, pharmaceutical products, personal care
products, dairy products or environmental samples. The analysis of
preferred beverages include soda, bottled water, fruit juice, beer,
wine or liquor products. Suitable PNA probes, probe sets, methods
and kits will be particularly useful for the analysis of raw
materials, equipment, products or processes used to manufacture or
store food, beverages, water, pharmaceutical products, personal
care products dairy products or environmental samples.
[0120] Additionally, the PNA probes, probe sets, methods and kits
of this invention are particularly useful for the detection of
bacteria and eucarya (e.g. pathogens) in clinical samples and
clinical environments. Suitable PNA probes, probe sets, methods and
kits will be particularly useful for the analysis of clinical
specimens, equipment, fixtures or products used to treat humans or
animals.
[0121] Having described the preferred embodiments of the invention,
it will now become apparent to one of skill in the art that other
embodiments incorporating the concepts described herein may be
used. It is felt, therefore, that these embodiments should not be
limited to disclosed embodiments but rather should be limited only
by the spirit and scope of the following claims.
EXAMPLES
[0122] This invention is now illustrated by the following examples
which are not intended to be limiting in any way.
Example 1
[0123] Synthesis of bis-(2-methoxyethyl)amidyl-diglycolic acid
[0124] To 500 mmol of diglycolic anhydride stirring in 800 mL of
dichloromethane (DCM) was added dropwise, 1.1 mole of
bis(2-methoxyethyl)amine (Aldrich Chemical). The reaction was
allowed to stir for 2 hours and then 280 mL of 6N HCl was added
dropwise. The contents were then transferred to a separatory funnel
and allowed to separate. The DCM layer was removed and the aqueous
layer extracted with 100 mL of DCM. The combined DCM layers were
then extracted with 100 mL of 10% aqueous citric acid. The DCM
layer was then separated, dried (Na.sub.2SO.sub.4), filtered and
evaporated to yield 73.8 g (296 mmole; 59 % yield). A kugelrorh was
then used to remove traces of solvent (product was heated to
60.degree. C. at approximately 180 .mu.M Hg).
Example 2
[0125] Synthesis of
N-[N"-Fmoc-(2"-aminoethyl)]-N-[N,N'-(2-methoxyethyl)am-
idyl-diglycolyl]glycine ("Fmoc-"E"aeg-OH")
[0126] To 60 mmol of Fmoc-aeg-OH (PerSeptive Biosystems, Inc.) was
added 360 mL of MilliQ water, 180 mL of acetone, 120 mmol of
NaHCO.sub.3 and 60 mmol of K.sub.2CO.sub.3. This solution was
allowed to stir until all the Fmoc-aeg-OH had dissolved (approx. 2
hr.) and then the solution prepared, as described below, was
added.
[0127] To 70 mmol of bis-(2-methoxyethyl)amidyl-diglycolic acid was
added 120 mL of anhydrous acetonitrile (Fluka Chemical), 240 mmol
of N-methylmorpholine (NMM; Fluka Chemical) and 75 mmol of
trimethylacetyl chloride (Aldrich Chemical). The solution was
allowed to stir at room temperature for 30 minutes and then added
dropwise to the solution of Fmoc-aeg-OH which was prepared as
described above.
[0128] After the combined solutions stirred for 1 hr and tlc
analysis indicated complete reaction, 6N HCl was added to the
reaction until the pH was less than 2 by paper. The organic solvent
was then removed by vacuum evaporation. The remaining aqueous
solution was then transferred to a separatory funnel and extracted
2.times. with 250 mL of ethyl acetate. The combined ethyl acetate
layers were dried (Na.sub.2SO.sub.4), filtered and evaporated to
yield 41.5 g of an oil.
[0129] This crude product was purified by column chromatography
using a reversed phase stationary phase (C18) and a gradient of
aqueous acetonitrile to elute the product and remove the pivalic
acid. Though not visible by tlc, the elution of the pivalic acid
can be monitored by smell. The pivalic acid can be almost
completely eluted from the column prior to elution of the product.
Elution of the product can be monitored by tlc. Yield 26.8g (47
mmol; 78%). This "Fmoc-"E"aeg-OH" monomer was used directly on the
PNA synthesis instrument, using standard condensation conditions,
or used to prepare prederivatized synthesis supports which were
used for the preparation of C-terminally "E" modified PNAs. An "E"
modification (subunit) of a PNA or polyamide has the formula: 2
Example 3
[0130] Synthesis of 4-(N-(tert-butyloxycarbonyl)-aminobenzoic
acid:
[0131] To 100 mM of methyl-4amino benzoic acid stirring in 150 mL
of dioxane was added 110 mM of di-tert-butyl-dicarbonate. The
reaction was warmed to 70-80.degree. C. and let stir for about 48
hours. The solvent was then evaporated under reduced pressure and
the residue redissolved in about 300 mL of ethylacetate. The
organic layer was then washed three times with 10% aqueous citric
acid, dried (Na.sub.2SO.sub.4), filtered and evaporated to a solid.
The solid was then suspended in 150 mL of 1N NaOH and 50 mL
acetone. Saponification of the ester was allowed to run overnight
until complete hydrolysis was observed by thin layer chromatography
(TLC). To the solution was added citric acid until the pH of the
solution was approximately 4. The solid was then collected by
vacuum filtration and dried in a vacuum oven at 50.degree. C. Yield
2.sup.0.3g, 85%. The product was a single peak when analyzed by
reversed phase High Performance Liquid Chromatography (HPLC) using
0.1% aqueous trifluoroacetic acid (TFA) and a linear acetonitrile
gradient.
Example 4
[0132] Synthesis of
N-.alpha.-(Fmoc)-N-.epsilon.-[4-(N-(tert-butyloxycarbo-
nyl)-aminobenzamidyl)]-L-Lysine-OH ("Fmoc-K(P)-OH")
[0133] To 2.6 mmol of
N-.alpha.-(Fmoc)-N-.epsilon.-(NH.sub.2)-L-Lysine-OH was added 5 mL
of N,N'-dimethylformamide (DMF) and 2.7 mmol of trifluoroacetic
acid. This solution was allowed to stir until the amino acid had
completely dissolved.
[0134] To 2.6 mmol of 4-(N-(tert-butyloxycarbonyl)-aminobenzoic
acid was added 50 mL of DMF, 2.7 mmole of
[O-(7-azabenzotriaol-1-yl)-1,1,3,3-tetra- methyluronium
hexafluorophosphate (HATU), and 15 mmol of diisopropylethylamine
(DIEA). To this stirring solution was added, dropwise, the
N-.alpha.-(Fmoc)-N-.epsilon.-(NH.sub.2)-L-Lysine-OH solution
prepared as described above. The reaction was allowed to stir for
30 minutes and was then worked up.
[0135] The solvent was vacuum evaporated and the residue
partitioned in 100 mL of DCM and 50 mL of 10% aqueous citric acid.
The layers were separated and the organic layer washed with aqueous
5 % sodium bicarbonate. The product crystallized in the separatory
funnel and was collected by vacuum filtration. The solid as stirred
in a solution of 30 mL 10% aqueous citric acid and 70 mL DCM and
then recollected by vacuum filtration. Yield 1.58 mmol, 60%.
Example 5
[0136] Synthesis of PNAs
[0137] Unless otherwise stated, PNAs were synthesized using
commercially available reagents and instrumentation obtained from
PE Biosystems, Foster City, Calif., USA. PNAs having modifications
of "E" or "K(P)" ("P" is aminobenzoic acid) were prepared using
prederivatized synthesis support or by performing the synthesis
using monomers prepared as described above.
Example 6
[0138] Synthesis of Arylamine Labeled Peptide Nucleic Acids:
[0139] a. N-terminal Labeling:
[0140] Labeling of the amino terminus of the PNA oligomer with a
linker group while the oligomer was still support bound was
accomplished by condensation of two subunits of Expedite PNA Linker
(P/N GEN063032) using one of the auxiliary positions of the PNA
synthesizer and the standard coupling cycle. To the amino terminus
of the elongated polymer was condensed
4-(N-(tert-butyloxycarbonyl)-aminobenzoic acid (See: Example 3).
Condensation of 4-(N-(tert-butyloxycarbonyl)-aminobenzoic acid with
the N-terminus was typically performed manually using conditions
similar to those used on the PNA synthesizer except that the
concentration of reagents and reaction time was usually increased.
After desired modification of the amino terminus of the polymer,
the oligomers were then cleaved from the support, deprotected and
purified using reversed phase HPLC.
[0141] b. C-terminal Labeling:
[0142] Bulk synthesis support having a prederviatized
N-.epsilon.-arylamine-L-lysine residue was prepared by condensing
N-.alpha.-(Fmoc)-N-.epsilon.-[4-(N-(tert-butyloxycarbonyl)-aminobenzamidy-
l)]-L-Lysine-OH ("Fmoc-K(P)-OH") with the PNA synthesis support
prior to assembly of the PNA. Condensation of the "Fmoc-K(P)-OH"
monomer with the synthesis support was typically performed manually
using conditions similar to those used on the PNA synthesizer
except that the concentration of reagents and reaction time was
typically increased. After desired PNA synthesis and
labeling/modification, C-terminal aryl amine modified oligomers
were then cleaved from the support, deprotected and purified using
reversed phase HPLC.
Example 7
[0143] Preferred Method For Removal Of The Fmoc Protecting
Group
[0144] The synthesis support was treated with a solution of 25%
piperidine in DMF for 10-15 minutes at room temperature. After
treatment, the synthesis support was washed and dried under high
vacuum. The support can then be treated with labeling reagent (See:
Examples 6 and 8).
Example 8
[0145] Preferred Method For Amine Labeling of Support Bound PNA
with the NHS esters of 5(6)carboxyfluorescein (Flu), or 5(and
6)-carboxy-X-rhodamine (Rox)
[0146] The amino protecting group (Fmoc) of the assembled PNA was
removed and the synthesis support was washed and dried under
vacuum. The synthesis support was then treated for 4-5 hours at
30-37.degree. C. with approximately 250 .mu.L of a solution
containing 0.08 M NHS ester labeling reagent, 0.24 M DIEA and 0.24
M 2,6-lutidine. After treatment the synthesis support was washed
and dried under high vacuum. The PNA oligomer was then cleaved,
deprotected and purified using reversed phase HPLC using methods
known in the art.
Example 9
[0147] General Procedure For Cleavage, Deprotection and
Purification
[0148] The synthesis support (Fmoc-PAL-PEG/PS; P/N GEN913384) was
then removed from the synthesis cartridge, transferred to a
Ultrafree spin cartridge (Millipore Corp., P/N SE3P230J3) and
treated with a solution of TFA/m-cresol (either of 7/3 or 8/2
(preferred)) for 1-3 hours. The solution was spun through the
support bed and again the support was treated with a solution of
TFA/m-cresol for 1-3 hours. The solution was again spun through the
support bed. The combined eluents (TFA/m-cresol) was then
precipitated by addition of approximately 1 mL of diethyl ether.
The precipitate was pelletized by centrifugation. The pellet was
then resuspended in ethyl ether and pelletized two additional
times. The dried pellet was then resuspended in 20% aqueous
acetonitrile (ACN) containing 0.1 % TFA (additional ACN was added
as necessary to dissolve the pellet). The product was analyzed and
purified using reversed phase chromatographic methods known in the
art.
[0149] Note: Several PNAs were prepared using new product
Fmoc-XAL-PEG/PS synthesis support (P/N GEN 913394) available from
PE Biosystems, Foster City, Calif., USA. This support has the
advantage that the PNA can be removed more rapidly and under more
mildly acid conditions. For PNAs prepared with Fmoc-XAL-PEG/PS the
support is treated as described above except that a solution of
TFA/m-cresol 9/1 was used for a period of 10-15 minutes
(2.times.).
Example 10
[0150] Exemplary Procedures For Preparing Peptide Nucleic Acids
(PNAs) Conjugated To Soybean Peroxidase
[0151] Stock Solutions:
[0152] 1. Probe Stock:
[0153] Purified arylamine terminated probe, typically fifteen or
sixteen residues in length, was dissolved at a concentration of
approximately 0.33 .mu.mol per milliliter in 50% aqueous
N,N'-dimethylformamide (DMF).
[0154] 2. Enzyme Stock:
[0155] Soy bean peroxidase, conjugate grade, obtained from Wiley
Organics, was dissolved at a concentration of 8.0 mg per milliliter
in an aqueous buffer comprised of 0.3 M NaCl, 10 mM MgCl.sub.2, 0.1
mM ZnCl.sub.2 and 30 mM N-methylmorpholine adjusted to pH 7.6 with
12 N hydrochloric acid.
[0156] 3. 30% Aqueous DMF:
[0157] An aqueous DMF solution was prepared by combining three
volumes of DMF with 7 volumes of water.
[0158] 4. MES Buffer
[0159] An 0.2 M solution of 4-morpholineethanesulfonic acid (MES)
in water was prepared (not pH adjusted).
[0160] 5. Glycine Solution
[0161] A solution comprised of 0.5 M glycine and 0.25 M sodium
hydroxide in water was prepared.
[0162] 6. Wash Buffer
[0163] An aqueous buffer comprised of 0.15 M NaCl, 5 mM MgCl.sub.2,
0.05mM ZnCl.sub.2 and 15 mM N-methylmorpholine adjusted to pH 7.6
with hydrochloric acid was prepared.
[0164] 7. Storage Buffer
[0165] An aqueous buffer comprised of 0.3 M NaCl, 10 mM MgCl.sub.2,
0.1 mM ZnCl.sub.2 and 30 mM N-methylmorpholine adjusted to pH 7.6
with 12 N hydrochloric acid was prepared.
[0166] 8. Stabilization Buffer
[0167] Peroxidase Stabilizing Buffer, DAKO Diagnostics Canada Inc.;
Part No. D210084
[0168] Exemplary Small Scale Conjugation Procedure:
[0169] Note: This procedure has been successfully scaled at least
10 fold.
[0170] In a reaction tube was combined 20 .mu.L of Enzyme Stock,
12.5 .mu.L of 30% Aqueous DMF, and 7 .mu.L of Probe Stock. In a
separate tube was placed 1 mg of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)
and 10 .mu.L of MES Buffer. These reagents were mixed, just prior
to addition to the reaction, until the EDC had dissolved in the MES
Buffer. The EDC/MES Buffer solution was then added to the tube
containing the enzyme and probe (Reaction Mixture). The contents
were mixed, and the tube was placed at 0.degree. C. for 40 min. To
the Reaction Mixture was then added 7 .mu.L of Glycine Solution.
The contents were again mixed and the tube was placed at 0.degree.
C. for a further 20 minutes.
[0171] Exemplary Conjugate Purification Procedures:
[0172] We have used both ultrafiltration and gel filtration
chromatography for the purification of the enzyme-linked probe from
excess enzyme and excess probe. At this time we prefer to use gel
filtration chromatography though these as well as other methods of
separation may work equally well. Exemplary methods for both
purification procedures will be described below.
[0173] Ultrafiltration
[0174] As an example of ultrafiltration, the contents of the tube
(See: Exemplary Small Scale Conjugation Procedure) were diluted
with 50 .mu.L of Wash Buffer and then transferred to the cup of an
ultrafiltration device (e.g. 30,000 molecular weight cut-off,
Millipore Corporation, Bedford Mass.) and spun at 5,000.times.g
until .about.90% of the liquid had been removed from the cup. An
additional 50 .mu.L of Wash Buffer was then be added to the cup and
the device spun again to remove 90% of the liquid. This washing
procedure was preferably repeated two additional times. The
contents of the cup were then diluted to a volume of 1 milliliter
in Storage Buffer. The absorbance of this solution (at 260 nm) was
then used to estimate the concentration of the enzyme conjugate
(0.05 absorbance units at 260 nanometers per milliliter is
typically estimated to be 0.33 nmol per milliliter based on an
estimated extinction for a PNA 15-mer of 150 optical density units
per .mu.mole of probe).
[0175] Gel Filtration Chromatography
[0176] At this time however, we prefer to separate the crude
reaction mixture using gel filtration chromoatography (e.g.
Superdex 200 (Part No. 17-1043-01) from Amersham Pharmacia or a
BioRad prepacked gel filtration BioSelect SEC 250-5 column (Part
No. 125-0476)). The mobile phase was aqueous 0.1M NaCl and 0.1M
bis-tris-HCl (Research Organics; Part No. 1164B) pH 6.5 with 10%
acetonitrile. After Gel Filtration Chromatography, the fractions
were desalted and resuspended in Stabilization Buffer (this buffer
is superior to the Storage Buffer for long term storage of the
conjugate). Desalting was performed by loading the fractions on a
preconditioned (See: Manufacturers instructions) Oasis.TM.
prepackaged column (Waters; Part No. 094225 (30 mg); 094226 (60 mg)
or 106202 (200 mg)). Once loaded, the enzyme conjugate was eluted
from the stationary phase using a solution containing 0.01 M NaCl,
0.02 M Tris pH 7.4 with a stepwise (10% per step) gradient of
aqueous acetonitrile (usually requiring 30% aqueous acetonitrile to
elute the product). The absorbance of this solution (at 260 nm) was
then used to estimate the concentration of the enzyme conjugate
(0.05 absorbance units at 260 nanometers per milliliter is
typically estimated to be 0.33 nmol per milliliter based on an
estimated extinction for a PNA 15-mer of 150 optical density units
per .mu.mole of probe). The aqueous acetonitrile was removed by
vacuum evaporation and the conjugate resuspended in Stabilization
Buffer based on the pre-evaporation quantity of conjugate in the
sample. Generally, the conjugate was resuspended to a concentration
of 10 .mu.M in Stabilization Buffer and stored at -20.degree. C.
The SBP probes can optionally be diluted to 100 nM in Stabilization
Buffer and stored at 4.degree. C.
[0177] Final Probe Preparation
[0178] Regardless of which purification method is used, the
products are typically screened using dot blot analysis on nylon
membrane (See Example 10) to determine sensitivity, specificity and
noise. Analysis was performed after the fractions were transferred
to Storage Buffer or Stabilization Buffer.
2TABLE 2 PNA Oligomers Prepared Probe Target No Probe ID Organism
PNA Probe Sequence 1 Flu-BacUni-1 Bacteria
Flu-OO-CTG-CCT-CCC-GTA-GGA-NH.sub.2 2 Flu-BacUni-2 Bacteria
Flu-OO-TAC-CAG-GGT-ATC-TAA-T-NH.sub.2 3 Flu-BacUni-3 Bacteria
Flu-OO-CAC-GAG-CTG-ACG-ACA-NH.sub.2 4 Flu-BacUni-4 Bacteria
Flu-OO-CCG-ACA-AGG-AAT-TTC-NH.sub.2 5 SBP-BacUni-1 Bacteria
SBP-P-OO-CTG-CCT-CCC-GTA-GGA-NH.sub.2 6 SBP-BacUni-2 Bacteria
SBP-P-OO-TAC-CAG-GGT-ATC-TAA-T-NH.sub.2 7 SBP-BacUni-3 Bacteria
SBP-P-OO-CAC-GAG-CTG-ACG-ACA-NH.sub.2 8 SBP-BacUni-4 Bacteria
SBP-P-OO-CCG-ACA-AGG-AAT-TTC-NH.sub.2 9 SBP/Flu-BacUni-1 Bacteria
Flu-OEE-CTG-CCT-CCC-GTA-GGA-EOO-K(P- SBP)-NH.sub.2 10 Flu-EuUni-1
Eucarya Flu-OO-ACC-AGA-CTT-GCC-CTC-C- -NH.sub.2 11 Flu-EuUni-2
Eucarya Flu-OO-GGG-CAT-CAC-AGA-CCT-G-NH.s- ub.2 12 Flu-EuUni-3
Eucarya Flu-OOE-TAG-AAA-GGG-CAG-GGA-EE-NH.sub.- 2 13 Flu-EuUni-4
Eucarya Flu-OEE-TAC-AAA-GGG-CAG-CCA-EE-NH.sub.2 14 SBP-EuUni-1
Eucarya SBP-P-OO-ACC-AGA-CTT-GCC-CTC-C-NH.sub.2 15 SBP-EuUni-2
Eucarya SBP-P-OO-GGG-CAT-CAC-AGA-CCT-G-NH.sub.2 16 Flu-BacUni-1EE
Bacteria Flu-OO-CTG-CCT-CCC-GTA-GGA-EE-NH.sub.2 17 Rox-EuUni-1EE-
Eucarya Rox-OO-CCA-GAC-TTG-CCC-TCC-EE-NH.sub.2 15mer
[0179] All PNA sequences are written from the amine (N-) terminus
to the carboxyl (C-) terminus. Flu=5(6)-carboxyfluorescein,
Rox=5(and 6)-carboxy-X-rhodamine; SBP=soy bean peroxidase;
P=4-aminobenzoic acid; K=L-lysine; E is defined above; and
O=8-amino-3,6-dixoactanoic acid.
Example 11
[0180] General Methods For Dot Blot Analysis:
[0181] RNA Preparation:
[0182] Using a Qiagen kit (P/N 75144), total RNA (including app.
80% rRNA) was isolated from the different bacteria or eucarya cells
which had been grown in culture. The total concentration of
isolated RNA was determined by measuring the absorption at 260
nm.
[0183] PNA Probe Hybridization to the Membranes:
[0184] Note: The text which has been bracketed and underlined was a
modification to the procedure which was used to generate the
results presented in FIGS. 4, 5 and 7 (optimized for SBP labeled
PNA probes). This is the preferred procedure as can be seen by the
improved signal to noise ratio when compared with the images
presented in FIG. 2.
[0185] Dot blots were made on nylon membranes obtained from
Gibco-BRL (P/N 14830-012). For the rRNA of each cultured bacteria
or eucarya, a dilution row containing at least 5 spots was made,
starting with a concentration of approximately 16 .mu.g/.mu.L RNA
for the strongest solution and continuing with half log dilutions
in diethyl pyrocarbonate (DEPC) treated water (RNase free). Prior
to spotting on the membrane, each dilution stock was heated to
68.degree. C. for three minutes. The spotting produced a half log
dilution series containing approximately 16, 5.1, 1.6, 0.52, 0.17
ng . . . (etc.) total RNA per spot. Once the spots had air dried,
the membrane was UV-crosslinked and then stored in a plastic bag
until used.
[0186] When used, individual membranes were placed in plastic bags
and pre-wet with RNase free water. The membranes were prehybridized
in Hybridization Buffer (20 mM Tris-HCl, pH 7.5; 50% formamide;
0.1% sodium dodecyl sulfate (SDS); and 100 mM NaCl) for 15 minutes
at 50.degree. C. [The membranes were prehybridized in Hybridization
Buffer 2 (20 mM Tris-HCl, pH 9.5; 50% formamide; 1.times.Denhardt's
Solution (USB, P/N US70468); 0.7% polyoxyethylene-sorbitan
monolaurate (Tween 20, Sigma P/N P-1379); and 100 mM NaCl) for 15
minutes at 50.degree. C.
[0187] All fluorescein labeled probes were diluted in 1:1
DMF/H.sub.2O to a concentration of approximately 300 pmole/.mu.L
and then diluted to a final concentration of 5 pmol/mL each using
Hybridization Buffer. All SBP labeled probes were prepared and
diluted as described in Example 10. The prehybridization buffer was
removed from the bags and fresh hybridization buffer containing the
PNA probe(s) of interest was(were) added to the bags. The
appropriate probe(s) was(were) typically used at a final
concentration of 5 pmol/mL each for Fluorescein labeled PNAs or 1
pmol/mL for SBP labeled PNAs (except for Panels I and II of FIG. 4,
wherein 5 pmol/mL of the bis labeled probe (SBP/Flu-BacUni-1) was
used for direct comparison).
[0188] Probe hybridization was performed at 50.degree. C. for 1
hour except that SBP probes were hybridized for 30 minutes. The
filters treated with Fluorescein labeled probes were then washed 3
times in TE-7.5 (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) containing 0.2%
SDS at ambient or elevated temperature. The filters treated with
SBP labeled probes were then washed 3 times in TE-9.0 (10 mM
Tris-HCl, pH 9.0, 1 mM EDTA) containing 0.2% Tween 20 at elevated
temperature. [The wash buffer was also modified whereby the 10 mM
Tris was replaced with 10 mM Capso (Sigma P/N C-1254) pH 10.]
[0189] RNA Spotted Membranes:
[0190] With reference to FIG. 1 (Panels I, II, III, IV and V), the
total-RNA of each of the following bacteria were spotted on
membranes in the columns illustrated: A Pseudomonas aeruginosa, B
Escherichia coli, C Staphylococcus aureus and D Salmonella
typhimurium.
[0191] With reference to FIG. 2 (Panels I, II, III and IV) and FIG.
4 (Panels I and II), the total-RNA of each of the following
bacteria were spotted on membranes in the columns illustrated: A
Pseudomonas fluorescens, B Pseudomonas aeruginosa, C Pseudomonas
cepatia, D Pseudomonas putida, E Escherichia coli, F Bacillus
subtilis, G Staphylococcus epidermidis, H Staphylococcus aureus,
and I Salmonella typhimurium.
[0192] With reference to FIG. 3 (Panels I, II, III and IV), the
total-RNA of each of the following organisms were spotted on
membranes in the columns illustrated: A Human spleen, B Mouse
spleen, C Rat Kidney, D Saccaharomyces cerevisiae, E Dekerra
brettanomyces and F E. coli.
[0193] With reference to FIG. 5 (Panels I and II) and FIG. 7
(Panels I and II), the total RNA of each of the following organisms
were spotted on membranes in the columns illustrated: A S.
cerevisiae, B Zygosaccaharomyces rouxii, C Dekerra intermedia, D
Zygosaccaharomyces balii, E Lactobacillus brevis and F Pediococcus
damnosus.
[0194] Visualization of the Membrane:
[0195] 1. For Fluorescein Labeled Probes:
[0196] After the washes were completed, the membranes were treated
with a blocking solution (50 mM Tris-HCl, pH 9.0; 0.5 M NaCl; and
0.5% casein). The starting temperature of the solution was
65.degree. C., but the solution cooled as the blocking proceeded
with shaking at room temperature for 15 minutes. An
anti-fluorescein-alkaline phosphatase conjugate (Rabbit (Fab)
anti-FITC/AP (DAKO A/S, P/N K0046)) was diluted 1:1000 in blocking
solution and the membranes were left shaking in this solution for
30 minutes at room temperature. The membranes were then washed in a
washing solution (50 mM Tris-HCl, pH 9.0; 0.5 M NaCl; and 0.5%
Tween-20) three times each for 5 minutes. To prepare the membranes
for the detection, a final rinse was performed (10 mM Tris-HCl, pH
9.5; 10 mM NaCl; and 1 mM MgCl2). The chemiluminescent substrate
(AMPPD, Tropix Corp., P/N PD025) was diluted 1:100 in an aqueous
Substrate Solution (0.1 M diethanolamine, pH 9.7; and 1 mM MgCl2)
and the membranes were immersed without shaking for 4 minutes. The
membranes were placed in a plastic bag and excess substrate was
squeezed out and the bag sealed. The membranes were exposed to
Fuji-RX X-ray film for between 5 and 30 minutes.
[0197] Note: The membranes used to generate FIG. 3 were treated as
Follows: Hybridization Buffer 2 was used as the modified wash
buffer. In addition, CDP* (Tropix P/N MS025) was used as the
substrate diluted 1:1000 in Substrate Solution.
[0198] 2. For SBP Labeled Probes:
[0199] After the washes were completed, the chemiluminescent
substrate (Pierce 17015) was mixed 1:1 and CaCl.sub.2 added to a
final concentration of 50 mM. The membranes were immersed in the
substrate without shaking for 2 minutes. The membranes were placed
in a plastic bag and excess substrate was squeezed out and the bag
sealed. The membranes were exposed to Fuji-RX X-ray film for
between 1 and 15 minutes.
[0200] Results:
[0201] With reference to FIG. 1, Panels I through V, the spotted
RNA of four species of bacteria was easily detected using each of
the individual the fluorescein labeled PNA probes (Probe Nos. 1, 2,
3 and 4) directed to bacteria (Panels II through V) and a mixture
of all four fluorescein labeled PNA probes (Probe Nos. 1, 2, 3, and
4; Panel I) directed to bacteria. The limit of detection was
visibly improved by approximately 1 log unit when using the PNA
probe mixture (Panel I). This data demonstrated the universal
nature of the fluorescein labeled PNA probes for the detection of
bacteria and the improvement in the limit of detectability when
using a mixture of several universal PNA probes.
[0202] With reference to FIG. 2, Panels I through IV, the spotted
RNA of eight species of bacteria was easily detected using each of
the SBP labeled PNA probes (Probe Nos. 5, 6, 7 and 8). This data
demonstrated the universal nature of the SBP labeled PNA probes for
the detection of bacteria. When taken as a whole, the data
presented in FIGS. 1 and 2 demonstrates that both PNA probes
labeled with either detectable moiety (SBP v. fluorescein) are
suitable for universal detection of target nucleic acid sequences
of bacteria.
[0203] With reference to FIG. 3, Panels I, II, III and IV the
spotted RNA of five species of multicellular and single cellular
organisms was easily detected using each of the individual the
fluorescein labeled PNA probes (Probe Nos., 10, 11, 12 and 13).
Probes 12 and 13 did yield a slightly detectable cross reaction to
the bacterial species (E. coli) but this was very mild as compared
with the specific signal detected in the presence of the rRNA of
the yeasts. Moreover, more stringent washing would likely result in
elimination of the non-specific signal. Curiously, the perfectly
complementary probe no. 13, exhibited less cross reaction as
compared with the less homologous probe no. 12. Nevertheless, this
data demonstrated the universal nature of the fluorescein labeled
PNA probes for the detection of eucarya. Similar results have been
obtained with SBP labeled PNA probes based on EuUni-1 and EuUni-2.
These results taken as a whole demonstrate that PNA probes labeled
with either detectable moiety (SBP v. fluorescein) are suitable for
universal detection of target nucleic acid sequences of
eucarya.
[0204] With reference to FIG. 4, Panels I and II, the spotted RNA
of all bacteria was easily detected using a PNA probe labeled with
both fluorescein and SBP (bis labeled PNA probe; Probe No. 9). This
data demonstrated the universal nature of the bis-labeled PNA probe
for the detection of the eight species of bacteria, as well as
providing a means to directly compare SBP and fluorescein labeled
PNA probes. The data indicates that while both labels are
detectable, detection of the SBP label provides greater sensitivity
and better signal to noise ratios under the conditions
examined.
[0205] With reference to FIG. 5 (Panels I and II), the spotted RNA
of either eucarya or bacteria was detected with the appropriate PNA
probe (for eucarya (Panel I), Probe No. 14/SBP-EuUni-1 was used;
and for bacteria (Panel II); Probe No. 5/SBP-BacUni-1 was used).
The data demonstrated that both probes exhibited very good
specificity for intended target organism while no significant cross
reaction was observed with the RNA of the non-target organisms.
[0206] With reference to FIG. 7 (Panels I and II), the spotted RNA
of either eucarya or bacteria was detected with the mixtures of the
appropriate PNA probe (for eucarya (Panel I), PNA probes Probe Nos.
14 and 15 were used; and for bacteria (Panel II); PNA probes Seq.
ID Nos. 5, 6, 7 and 8 were used). The data demonstrated that both
probes exhibited very good specificity for intended target organism
while no significant cross reaction was observed with the RNA of
the non-target organisms.
Example 12
[0207] Multiplex PNA-FISH
[0208] A 3 mL culture of E. coli was grown overnight in Tryptic Soy
Broth (TSB) at 30 .degree. C. Absorbance at 600 nm was measured and
the culture was diluted into fresh TSB until the absorbance at 600
nm was 0.5 OD/mL. This diluted culture stock was then allowed to
double 3-4 times before harvesting. Cells from a 20 mL culture were
pelleted by centrifugation at 8000 rpm for 5 minutes, resuspended
in 20 mL PBS (7 mM Na.sub.2HPO.sub.4; 3 mM NaH.sub.2PO.sub.4; 130
mM NaCl), pelleted again and resuspended in Fixation Buffer (3%
paraformaldehyde in PBS). The bacteria were incubated at room
temperature for 30-60 minutes before they were pelleted again
(centrifugation at 8000 rpm for 5 minutes). After removal of the
fixation solution, the cells were resuspended in 20 mL of 50 %
aqueous ethanol. The fixed bacteria may be used after 30 minutes of
incubation or stored at -20.degree. C. for up to several weeks
before being used.
[0209] Similarly, a 3 mL culture of Saccharomyces cerevisiae was
grown overnight in YM (Difco: DF 0711-17-1) at 25.degree. C.
Absorbance at 600 nm was measured and the culture was diluted into
fresh YM until the absorbance at 600 nm was 0.5 OD/mL. This diluted
culture stock was then allowed to double 3-4 times before
harvesting. Cells from a 20 mL culture were fixed as described
above.
[0210] To 100 .mu.L of each of these two preparations of fixed
cells (E. coli and Saccharomyces cerevisiae) in 50% aqueous ethanol
were mixed, centrifuged at 10,000 R.P.M. for 4 min. The aqueous
ethanol was then removed from the sample and the pellet was
resuspended in 100.mu.L of sterile PBS and pelleted again by
centrifugation at 10,000 R.P.M. for 4 min.
[0211] The PBS was then removed from the pellet, and the cells were
resuspended in 100 .mu.L of hybridization buffer (20 mM Tris-HCl,
pH 9.0; 100 mM NaCl; 0.5 % SDS) which contained the appropriate
probe (Flu-BacUni-1 EE (Probe No. 15) and Rox-EuUni-1 EE (Probe No.
16)) each at a concentration of 30 pmol/mL. The hybridization was
performed at 55.degree. C. for 30 minutes.
[0212] The sample was then centrifuged at 10,000 R.P.M. for 4 min.
The hybridization buffer was removed and the cells resuspended in
500 .mu.L sterile TE-9.0 (10 mM Tris-HCl, pH 9.0; 1 mM EDTA). The
solution was allowed to stand at 55.degree. C. for 10 minutes. The
sample was then centrifuged at 10,000 R.P.M. for 10 min. The TE-9.0
was removed from the pellet. This TE-9.0 wash was repeated two more
times.
[0213] After the final wash, the cells were resuspended in 100
.mu.L TE-9.0. An aliquot of 2 .mu.L of this suspension of cells was
placed on a glass slide, spread and allowed to dry. Next, 1-2 .mu.L
of Vectashield (Vector Laboratories, P/N H-1000) was deposited over
the dried cells and a coverslip was added to the slide and its
position fixed using a couple of drops of nail polish.
[0214] The bacteria were then observed using a Nikon fluorescent
microscope equipped with a 60.times.immersion oil objective, a
10.times.ocular (total enlargement is 600 fold) and light filters
obtained from Omega Optical (XF22 (green), XF34 (red)) and from
Chroma Technology Corp. (Triple filter (DAPI/FITC/TRITC)).
Electronic digital images were made of the slide using a SPOT
CCD-camera and software obtained from Diagnostic Instruments, Inc.,
Sterling Heights, MI (USA).
[0215] The digital images obtained, all covering the same section
of the slide, are presented in FIG. 6-I through 6-IV. In FIG. 6-I
(red image), yeast cells are stained red by the Rox-EuUni-1 EE
(probe no. 17). In FIG. 6-II (green image), E. coli is stained
green by the Flu-BacUni-1 EE (probe no. 16). In FIG. 6-III (triple
color image) both yeast and bacteria are visualized as a green
image as the field has been photographed using the cameras green
filter. Clearly the distribution of yeast and bacteria cells are
seen as a visual composite of the images presented in FIG. 6-I and
6-II. In FIG. 6-IV, a digitally created composite of the red and
the green images from FIG. 6-I and 6-II is presented for comparison
with the camera images presented in Panels I, II and III. These
figures clearly demonstrate that simultaneous detection of a
eucarya (represented by S. cerevisiea) and a bacteria (represented
by E. coli) in the same sample (multiplexing) is possible using PNA
probes. Though the multiplex analysis described herein is performed
manually by visual inspection of the digital images, software is
available for performing such a comparison to thereby generate
quantitative data for each of the target organisms present in the
sample.
Example 13
[0216] Rapid Identification and Enumeration of Colonies of Bacteria
and Yeast
[0217] I. Preparation of Yeast and Bacteria:
[0218] An overnight culture of E. coli was grown to produce the
bacteria for this Example. Similarly, an overnight culture of S.
cerevisiae was grown to produce the yeast for this Example. From
the bacteria culture, three sequential 100 fold dilutions were made
using 1.times.PBS as the diluent. From the yeast culture, two
sequential 100 fold dilutions were made using PBS as the diluent.
To prepare an exemplary sample, 500 .mu.L of each of the final
dilutions of the bacteria and the yeast were added to 50 mL of PBS.
A 5 mL aliquot of this exemplary sample, containing yeast and
bacteria, was then filtered through a 0.45.mu. PVDF membrane
(Millipore; P/N HVLP04700). This filtration was repeated to prepare
several membrane filters which could be used to analyze the E. coli
and S. cerevisiae present in the exemplary sample.
[0219] II. Membrane-based in-situ hybridization:
[0220] Membranes were aseptically transferred to a petripad soaked
with 2 mL of YM-medium (Sigma P/N L3022) in a small petridish and
incubated between 1.5 and 18 hours at 30.degree. C. E. coli
required as little as 1-2 hours of incubation at 37.degree. C.
before detectable micro-colonies were visible (data not shown).
However, the slower growing S. cerevisiae yeast required an
overnight culture (approximately 18 hours) before the
micro-colonies were clearly visible using the probes and methods
described herein.
[0221] Prior to hybridization with PNA probe(s), micro-colonies
were fixed to the membrane by placing the membrane on another
petripad soaked with 1.5 mL of Fixation Solution (0.5%
glutaraldehyde in ethanol with 10% Peroxidase Blocking Reagent;
DAKO P/N S2001) for 5 minutes. Probe hybridization was performed
for 30 minutes at 50.degree. C. in a petrislide (Millipore; P/N
PDMA 04700) with cover using 5 nM each of one or two SBP-labeled
PNA probes (Probe No. 5 for detection of bacteria and Probe No. 14
for detection of eucarya) in Hybridization Buffer 3 (25 mM
Tris-HCl, pH 9.5), 1.times.Denhardt's solution, 50% (v/v)
formamide, 0.7% (v/v) Tween 20, 1% Casein, 0.1 M NaCl, 5 mM EDTA).
Excess probe was removed by washing the filters for four times
seven minutes in wash solution (10 rnM CAPSO (pH 10.0), 0.2% (v/v)
Tween 20). Hybridized probe was then visualized by a two minute
chemiluminescent reaction using 500 .mu.L substrate (SuperSignal,
Pierce) followed by a 15 minutes exposure on X-ray film (Fuji).
[0222] With reference to FIGS. 8I-8III, the three membrane filters
(appearing in Panels I, II and III) were all grown for
approximately 18 hours and then treated with individual PNA probes
or a mixture of Probe No. 5 (for detection of bacteria) and Probe
No. 14 (for detection of eucarya). In panel I, only Probe No. 14
was used in the hybridization. The colonies observed were small
because the yeast grows slowly. The small spots represent colony
forming units of S. cerevisiae present in the volume of the
exemplary sample which was filtered onto the membrane. In panel
III, only Probe No. 5 was used in the hybridization. The colonies
observed were very large because the bacteria grow rapidly to form
large colonies within 18 hours. Each large spot represents a colony
forming unit of E. coli present in the volume of the exemplary
sample which was filtered onto the membrane. In panel II, a mixture
of both Probe No. 5 and Probe No. 14 was used in the hybridization.
Consequently, small colonies of S. cerevisiae and large colonies of
E. coli are visible in the Figure wherein the spots represent
colony forming units present in the volume of the exemplary sample
which was filtered onto the membrane. Taken as a whole, this
Example demonstrates the feasibility of multiplex culture analysis
of bacteria and eucarya using target specific probes comprising
identical labels wherein differentiation is based on culture
characteristics (growth rate or morphology) of the organism.
[0223] EQUIVALENTS
[0224] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those of ordinary skill in the art that various
changes in form and details may be made therein without departing
from the spirit and scope of the invention as defined by the
appended claims. Those skilled in the art will be able to
ascertain, using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed in the
scope of the claims.
Sequence CWU 1
1
8 1 15 DNA Artificial Sequence Description of Combined DNA/RNA
MoleculePNA Probing Nucleobase Sequence 1 ctgcctcccg tagga 15 2 16
DNA Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 2 taccagggta tctaat 16 3 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 3 cacgagctga cgaca 15 4 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 4 ccgacaagga atttc 15 5 16 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 5 accagacttg ccctcc 16 6 16 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 6 gggcatcaca gacctg 16 7 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 7 tagaaagggc aggga 15 8 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 8 tacaaagggc aggga 15
* * * * *