U.S. patent application number 10/631289 was filed with the patent office on 2006-01-05 for method for multiplex pna-fish.
Invention is credited to James M. Coull, Jens J. Hyldig-Nielsen.
Application Number | 20060003332 10/631289 |
Document ID | / |
Family ID | 29714557 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060003332 |
Kind Code |
A1 |
Hyldig-Nielsen; Jens J. ; et
al. |
January 5, 2006 |
Method for multiplex PNA-FISH
Abstract
This invention is related to multiplex PNA-FISH assays.
Inventors: |
Hyldig-Nielsen; Jens J.;
(Holliston, MA) ; Coull; James M.; (Westford,
MA) |
Correspondence
Address: |
APPLIED BIOSYSTEMS
500 OLD CONNECTICUT PATH
FRAMINGHAM
MA
01701
US
|
Family ID: |
29714557 |
Appl. No.: |
10/631289 |
Filed: |
July 31, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09335629 |
Jun 18, 1999 |
6664045 |
|
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10631289 |
Jul 31, 2003 |
|
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60089737 |
Jun 18, 1998 |
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Current U.S.
Class: |
435/6.1 ;
435/6.18; 530/350 |
Current CPC
Class: |
C07K 14/31 20130101;
C07K 14/21 20130101; Y02A 50/30 20180101; C07K 14/32 20130101; Y02A
50/451 20180101; C07K 14/255 20130101; C07H 21/00 20130101; C07K
14/003 20130101; C12Q 1/689 20130101 |
Class at
Publication: |
435/006 ;
530/350 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07K 14/00 20060101 C07K014/00 |
Claims
1. A multiplex PNA-FISH comprising: a) contacting a sample with two
or more fluorescently labeled independently detectable PNA probes,
wherein each of the two or more fluorescently labeled independently
detectable PNA probes comprise a probing nucleobase sequence that
hybridizes to a target sequence of one of the two or more organisms
of interest; and b) detecting, identify or quantitating
hybridization of the probing nucleobase sequence of the PNA probe
to the target sequence of the organism and correlating the result
for each of the two or more fluorescently labeled independently
detectable PNA probes with the presence, absence or number of the
two or more organisms of interest in the sample.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/335,629 filed on Jun. 18, 1999, which
application claims the benefit of U.S. Provisional Patent
Application Ser. No. 60/089,737 filed on Jun. 18, 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 microorganisms. More
specifically, this invention relates to a method for muliplex
PNA-FISH.
[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 has been particularly
elusive.
[0007] 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)).
[0008] 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,
O. 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, ln. 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)).
However, problems associated with solubility and self-aggregation
have recently been reduced or eliminated (See: Gildea et al., Tett.
Lett. 39: 7255-7258 (1998)). Nevertheless, their unique properties
clearly demonstrate that PNA is not the equivalent of a nucleic
acid in either structure or function. Consequently, 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.
[0009] In summary, 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 of microoganisms such as bacteria.
SUMMARY OF THE INVENTION
[0010] This invention is directed to PNA probes, probe sets,
methods and kits useful for detecting, identifying and/or
quantitating one or more organisms of interest in a sample wherein
the organisms are members of the bacterial species of E. coli,
Staphylococcus aureus, Pseudomonas aeruginosa, Pseudomonas cepatia,
Pseudomonas fluorescens or organisms of a bacterial genus including
the Salmonella genus, Bacillus genus or Pseudomonas genus. The PNA
probes and probe sets of this invention comprise probing nucleobase
sequences which allow for the specific detection of bacteria of a
target species or genus.
[0011] The preferred probing nucleobase sequence of the 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 certain species of bacteria,
certain genus of bacteria or members of a defined set of species
and genus of bacteria. In a most preferred embodiment, the probe
set is suitable for the detection, identification and/or
quantitation of USP bacteria (as defined herein).
[0012] This invention is further directed to a method suitable for
detecting, identifying and/or quantitating one or more organisms of
interest in a sample wherein the organisms are members of the
bacterial species of E. coli, Staphylococcus aureus, Pseudomonas
aeruginosa, Pseudomonas cepatia, Pseudomonas fluorescens or
organisms of a bacterial genus including the Salmonella genus,
Bacillus genus or Pseudomonas genus. The method comprises
contacting the sample with one or more PNA probes, wherein suitable
probes are described herein. According to the method, the presence,
absence or number of the one or more organisms of interest in the
sample are then detected, identified or quantitated. Detection,
identification and or quantitation is made possible by correlating
the hybridization, under suitable hybridization conditions or
suitable in-situ hybridization conditions, of the probing
nucleobase sequence of a PNA probe to the target sequence with the
presence, absence or number of the target organism of interest in
the sample. This correlation is made possible by direct or indirect
detection of the probe/target sequence hybrid.
[0013] In yet another embodiment, this invention is directed to
kits suitable for performing an assay which detects the presence,
absence or number of one or more organisms of interest in a sample.
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.
[0014] The PNA probes, probe sets, methods and kits of this
invention have been demonstrated to be highly specific for the
target organism(s) for which they are intended to detect. Moreover,
the assays described herein are rapid (2-3 hours or less),
sensitive, reliable and capable of both identification as well as
enumeration of organisms listed in Table 1 in a single assay. Since
probe-based analysis generically detects nucleic acid, 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.
[0015] This invention is also directed to a multiplex
PNA-fluorescent in-situ hybridization (FISH) assay. As a
demonstration of the versatility of the PNA probes, probe sets,
methods and kits of this invention, a PNA-FISH assay was
multiplexed without any change to the protocol. The analysis was
rapid, sensitive and reliable despite the substantial sequence
variations of the probing nucleobase sequence of the PNA probes
used for the different target organisms. Thus, Applicants have
demonstrated (believed to be the first successful example) the
feasibility of a multiplex PNA-FISH assay which can positively
detect, identify and quantitate two or more target organisms in a
single assay. Specifically, the multiplex assay as described in
Example 10 provided a means for the detection, identification and
quantitation of four target organisms using only three
independently detectable moieties (fluorophores).
[0016] The PNA probes, probe sets, methods and kits of this
invention are particularly useful for the detection of bacteria
(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.
[0017] Additionally, the PNA probes, probe sets, methods and kits
of this invention are particularly useful for the detection of
bacteria (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
[0018] FIGS. 1-I through 1-V are electronic images of dot blot
assays used to examine the specificity of PNA oligomers for certain
target organisms of a bacterial species.
[0019] FIGS. 2-I through 2-IV are electronic images of dot blot
assays used to examine the specificity of PNA oligomers for certain
target organisms of a bacterial genus.
[0020] FIGS. 3I through 3 IV are individual or composite digital
images of the same section of a sample slide of bacteria hybridized
with PNA probes wherein the PNA probes are independently
detectable. The images were obtained using a fluorescent microscope
and three different commercially available light filters. FIG. 3I
is the image obtained using a Green filter; Image 3-II is the image
obtained using a Red filter, FIG. 3-III is the image obtained using
a Blue filter and FIG. 3-IV is a digital composite of Images 3-II
and 3-III.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions:
[0021] 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.
[0022] 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.
[0023] c. As used herein, the term "target sequence" is the nucleic
acid nucleobase sequence of a specific bacteria which is to be
detected in an assay and to which at least a portion of the probing
nucleobase sequence of the bacteria specific probe is designed to
hybridize.
[0024] 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. No. 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) and
Lagriffoule et al., Chem. Eur. J., 3: 912-919 (1997).
[0025] In preferred embodiments, a PNA is a polymer comprising two
or more subunits of the formula: ##STR1## 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 integer from one to five. The integer t is 1 or 2 and the
integer 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.
[0026] 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.
[0027] 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.
[0028] 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.
2. Description
I. General:
PNA Synthesis:
[0029] Methods for the chemical assembly of PNAs are well known
(See: U.S. Pat. No. 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.
PNA Labeling:
[0030] Preferred non-limiting methods for labeling PNAs are
described in WO98/24933, WO99/22018 WO99/21881, the examples
section of this specification or are otherwise well known in the
art of PNA synthesis.
Labels:
[0031] 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.
[0032] Preferred haptens include 5(6)-carboxyfluorescein,
2,4-dinitrophenyl, digoxigenin, and biotin.
[0033] Preferred fluorochromes (fluorophores) include
5(6)-carboxyfluorescein (Flu),
6-((7-amino-4-methylcoumarin-3-acetyl)amino)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, Oreg.).
[0034] Preferred enzymes include polymerases (e.g. Taq polymerase,
Klenow PNA polymerase, T7 DNA polymerase, Sequenase, DNA polymerase
1 and phi29 polymerase), alkaline phosphatase (AP), horseradish
peroxidase (HRP) and most preferably, soy bean peroxidase
(SBP).
Detectable and Independently Detectable Moieties/Multiplex
Analysis:
[0035] 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
nucleic acid 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.
Spacer/Linker Moieties:
[0036] 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).sub.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.
Hybridization Conditions/Stringency:
[0037] 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.
Suitable Hybridization Conditions:
[0038] 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 comprise conditions suitable for performing an in-situ
hybridization procedure. Thus, suitable in-situ hybridization
conditions will become apparent using the disclosure provided
herein; with or without additional routine experimentation.
Blocking Probes:
[0039] 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.
Probing Nucleobase Sequence:
[0040] 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 wherein the presence,
absence or amount of target sequence can 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 hybridization conditions or suitable
in-situ hybridization conditions.
[0041] The probing nucleobase sequence suitable for detecting the
target organism listed in the table, will generally, but not
necessarily, have a length of 15 or fewer PNA subunits (most
preferably 7-15 subunits in length) wherein the exact nucleobase
sequence is at least 90% homologous to the probing 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 15-mer probing nucleobase
sequences are listed in Table 1. These probing nucleobase sequences
have been shown to be highly specific for the target organism in
the presence of the other organisms listed in the table.
[0042] The probing nucleobase sequence of a PNA probe will
generally have a probing nucleobase sequence which is 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)).
[0043] 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.
Probe Complexes:
[0044] 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 half or approximately half of the
complete target sequence of the bacteria 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.
II. Preferred Embodiments of the Invention:
a. PNA Probes:
[0045] In one embodiment, this invention is directed to PNA probes.
The PNA probes of this invention are suitable for detecting,
identifying or quantitating one or more organisms of interest in a
sample wherein the organisms are members of the bacterial species
of E. coli, Staphylococcus aureus, Pseudomonas aeruginosa,
Pseudomonas cepatia, Pseudomonas fluorescens or organisms of a
bacterial genus including the Salmonella genus, Bacillus genus or
Pseudomonas genus. General characteristics (e.g. length, labels,
nucleobase sequences, linkers etc.) of PNA probes suitable for the
detection, identification or quantitation of these specific
microorganisms or bacteria of a genus have been previously
described herein. The preferred probing nucleobase sequences of PNA
probes of this invention are listed in Table 1. TABLE-US-00001
TABLE 1 Probing Nucleobase Sequence Probe Target Probing ID# Type
Organism Nucleobase Sequence 1 Species E. coli TCA-ATG-AGC-AAA-GGT
2 Species S. aureus GCT-TCT-CGT-CCG-TTC 3 Species P.
CTG-AAT-CCA-GGA-GCA aeruginosa 4 AAC-TTG-CTG-AAC-CAC 5 Species P.
cepatia CCA-TCG-CAT-CTA-ACA 6 Species P. TCT-AGT-CAG-TCA-GTT
fluorescens 7 Genus Pseudomonas GCT-GGC-CTA-GCC-TTC 8
GTC-CTC-CTT-GCG-GTT 9 TTC-TCA-TCC-GCT-CGA 10 Genus Salmonella
CCG-ACT-TGA-CAG-ACC 11 CCT-GCC-AGT-TTC-GAA 12 Genus Bacillus
CTT-TGT-TCT-GTC-CAT Note: The target sequence to which these
probing nucleobase sequences hybridize have been analyzed using
sequence alignment analysis of information currently in Genbank
version 104. The alignment information indicates the sequences are
specific to the target organisms listed. However, the specificity
of probes must be functionally examined since sequence alignment
analysis does not always produce a target specific probe. The
probing nucleobase sequences listed above have been # determined to
be organism specific by actual screening methods.
[0046] 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 two or more
independently detectable moieties. Preferred independently
detectable moieties are independently detectable fluorophores.
[0047] 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.
[0048] 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 and 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 a bacteria of interest.
Unlabeled Non-Nucleic Acid Probes:
[0049] The probes of this invention need not be labeled with a
detectable moiety to be operable within the method of 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 and methods for
preparation and use are described in WIPO Patent Application
WO95/17430 and U.S. Pat. No. 5,612,458, herein incorporated by
reference.
[0050] 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 such as an enzyme. 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.
Advantages of Using PNA Probes
[0051] Fuchs et al. (Applied and Environmental Microbiology, 64:
4973-4982 (1998)) have demonstrated that the ability of nucleic
acid probes to hybridize to target sequences of rRNA (e.g. targets
like 16S or 23S rRNA) is highly dependent upon whether the site of
hybridization is placed in or outside the highly structured helix
regions. Moving the probe just a few bases in or out of such a
structured region can give rise to significant changes in the
overall signal intensity. The lack of signal intensity is believed
to result from the lack of probe accessibility to the hybridization
site within the secondary structure.
[0052] When designing species specific nucleic acid probes,
nucleobase sequence selection is further limited because rRNA is
relatively well conserved between related species. Moreover, the
limited number of species specific sequence variations are often
concentrated in the highly structured regions of the rRNA.
Therefore, some of the most useful regions of diverse nucleobase
sequences suitable for designing species specific probes are often
unavailable to nucleic acid probes.
[0053] Because of its unique structure, PNA probes can be designed
to target highly structured regions of rRNA. Thus, PNA probes do
not suffer from the limitations characteristic for nucleic acid
probes. For example, Salmonella 16S rRNA is less than 4% different
as compared with E. coli 16S rRNA. However, in the highly
structured helix #18 there exists a significant number of
differences (i.e. 14 nucleobase differences over a stretch of 26
nucleobases). This region would therefore be a fertile source of
variable sequence with which to attempt to design probes capable of
distinguishing between these two species. However, as reported by
Fuchs et al., DNA probes directed to this particular highly
structured region (Eco455 and Eco468 in Table 1 of Fuchs et al.)
generated only a small fraction (i.e. 3%) of the signal which was
obtained with the best DNA probe (Eco1482 in Table 1 of Fuchs et
al.). By comparison, a PNA probe directed to this structured region
(Probing Nucleobase Sequence 1 described in Table 1 of this
invention) functions well as determined by the signal obtained in
FIG. 3.
b. PNA Probe Sets:
[0054] In another embodiment, this invention is directed to a PNA
probe set suitable for detecting, identifying or quantitating one
or more organisms of interest in a sample wherein the organisms are
members of the bacterial species of E. coli, Staphylococcus aureus,
Pseudomonas aeruginosa, Pseudomonas cepatia, Pseudomonas
fluorescens or organisms of a bacterial genus including the
Salmonella genus, Bacillus genus or Pseudomonas genus. The general
and preferred characteristics of PNA probes suitable for the
detection, identification or quantitation of these specific
microorganisms or bacteria of a genus have been previously
described herein. Preferred probing nucleobase sequences are listed
in Table 1. The grouping of PNA probes within sets characterized
for specific groups of organisms (e.g. classification within
species or genus, etc.) is contemplated as a preferred embodiment
of this invention. 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 but preferably the blocking probes
are PNA.
[0055] Table 1 lists several species or genus of bacteria for which
two or more probing nucleobase sequences can be used to detect the
target organism. Where alternative probing nucleobase sequences
exist, it is preferable to use a probe set containing the two or
more PNA probes to thereby increase the detectable signal in the
assay.
[0056] One exemplary probe set would comprise probes suitable for
the detection of one or more bacterial species consisting of at
least E. coli, Staphylococcus aureus, Pseudomonas aeruginosa,
Pseudomonas cepatia and Pseudomonas fluorescens. A suitable probe
set would contain at least one, and preferably all, of the
following PNA probes. At least one PNA probe suitable for detecting
E. coli and having a probing nucleobase sequence
TCA-ATG-AGC-AAA-GGT. At least one PNA probe suitable for detecting
Staphylococcus aureus and having a probing nucleobase sequence
GCT-TCT-CGT-CCG-TTC. At least one PNA probe suitable for detecting
Pseudomonas aeruginosa and having a probing nucleobase sequence
selected from the group consisting of CTG-AAT-CCA-GGA-GCA and
AAC-TTG-CTG-AAC-CAC. At least one PNA probe suitable for detecting
Pseudomonas cepatia and having a probing nucleobase sequence
CCA-TCG-CAT-CTA-ACA. At least one PNA probe suitable for detecting
Pseudomonas fluorescens and having a probing nucleobase sequence
TCT-AGT-CAG-TCA-GTT. As previously suggested, the probe set may
contain other nucleic acid or PNA probes directed to other target
organisms or as blocking probes.
[0057] A second exemplary probe set would comprise probes suitable
for the detection of one or more bacteria of a genus consisting of
at least the Salmonella genus, Bacillus genus or Pseudomonas genus.
A suitable probe set would contain at least one, and preferably
all, of the following PNA probes. At least one PNA probe suitable
for detecting bacteria of the Salmonella genus and having a probing
nucleobase sequence selected from the group consisting of
CCG-ACT-TGA-CAG-ACC and CCT-GCC-AGT-TTC-GAA. At least one PNA probe
suitable for detecting bacteria of the Bacillus genus and having a
probing nucleobase sequence CTT-TGT-TCT-GTC-CAT. At least one PNA
probe suitable for detecting bacteria of the Pseudomonas genus and
having a probing nucleobase sequence selected from the group
consisting of GCT-GGC-CTA-GCC-TTC, GTC-CTC-CTT-GCG-GTT and
TTC-TCA-TCC-GCT-CGA. The three aforementioned PNA probing
nucleobase sequences have been found to be suitable for
specifically detecting bacteria of the Pseudomonas genus including
Pseudomonas aeruginosa, Pseudomonas cepatia, Pseudomonas
fluorescens and Pseudomonas putida. As previously suggested, the
probe set may contain other nucleic acid or PNA probes directed to
other target organisms or as blocking probes.
[0058] The detection, identification or quantitation of certain
organisms is particularly useful where these organisms comprise a
group of pathogens for which a contamination limit applies by
industry standard or by governmental regulation. One such group of
organisms are the bacterial pathogens of the United States
Pharmacopoeia (USP bacteria). USP bacteria include E. coli, the
Salmonella genus, Pseudomonas aeruginosa and Staphylococcus aureus.
Food, beverage and pharmaceutical products are routinely examined
for the presence, absence or number of these USP bacteria.
Consequently, a set of PNA probes suitable for the detection of the
USP bacteria is a preferred embodiment of this invention.
[0059] The probing nucleobase sequences of PNA probes suitable for
the detection of USP bacteria have been previously listed in Table
1. Most preferably, this set of PNA probes is suitable for the
detection of all of the USP bacteria. An exemplary probe set would
contain at least one, and preferably all, of the following PNA
probes. A PNA probe suitable for detecting E. coli and having a
probing nucleobase sequence TCA-ATG-AGC-AAA-GGT. At least one PNA
probe suitable for detecting Salmonella and having a probing
nucleobase sequence selected from the group consisting of
CCG-ACT-TGA-CAG-ACC and CCT-GCC-AGT-TTC-GAA. At least one PNA probe
suitable for detecting Pseudomonas aeruginosa and having a probing
nucleobase sequence selected from the group consisting of
CTG-AAT-CCA-GGA-GCA and AAC-TTG-CTG-AAC-CAC. A PNA probe suitable
for detecting Staphylococcus aureus and having a probing nucleobase
sequence GCT-TCT-CGT-CCG-TTC. As previously suggested, the probe
set may contain other nucleic acid or PNA probes directed to other
target organisms or as blocking probes.
c. Methods:
[0060] In another embodiment, this invention is directed to a
method suitable for detecting, identifying or quantitating one or
more organisms of interest in a sample wherein the organisms are
members of the bacterial species of E. coli, Staphylococcus aureus,
Pseudomonas aeruginosa, Pseudomonas cepatia, Pseudomonas
fluorescens or organisms of a bacterial genus including the
Salmonella genus, Bacillus genus or Pseudomonas genus. The general
and preferred characteristics of PNA probes suitable for the
detection, identification or quantitation of these target organisms
have been previously described herein. Preferred probing nucleobase
sequences are listed in Table 1.
[0061] The method comprises contacting the sample with one or more
PNA probes, wherein suitable probes have been previously described
herein. According to the method, the presence, absence or number of
the one or more organisms of interest in the sample are then
detected, identified or quantitated by correlating hybridization of
the probing nucleobase sequence of a PNA probe to the target
sequence of a target organism under suitable hybridization
conditions or suitable in-situ hybridization conditions.
[0062] The grouping of PNA probes within probe sets to be used with
methods for detecting specific groups of organisms (e.g.
classification within species, genus or USP bacteria) is
contemplated as a preferred embodiment of this invention. Exemplary
probes and probe sets suitable for the practice of this method have
been previously described herein.
Exemplary Media Based Analysis of Bacteria
[0063] The methods, kits and compositions of this invention are
particularly useful for the rapid probe-based detection,
identification and quantitation of viable bacteria. For example, it
is possible to use of enzyme-linked PNA probes in combination with
in-situ analysis of microcolonies of bacteria grown (using
selective media) directly on the medium on which they were isolated
from the sample (i.e. a filtration membrane) to thereby achieve the
rapid, sensitive and specific analysis of bacteria in a manner
which was not previously possible.
[0064] The rapid probe-based analysis of growing bacteria requires
very high sensitivity in addition to probe specificity because the
cell count is limited during the early stages of cell division.
Since probe-based analysis detects nucleic acid, the analysis of
growing bacteria is used to distinguish between viable organisms
and dead (non-viable) organisms, the presence of which are not
considered to cause food or beverage spoilage or contamination.
[0065] Enzyme-linked probes are preferred for such rapid 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.
[0066] In preferred embodiments, the bacteria are grown directly on
an isolation medium. Integration of the isolation medium with the
growth of the bacteria eliminates the need for a transfer pre- and
post-culture growth 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 bacteria will not pass though the pores thereby
insuring that all the bacteria in the sample is collected on the
filter.
[0067] Once the sample is collected on the isolation medium, the
bacteria are grown in a manner specific for the organism or
organisms of interest using methods known in the art. Preferably,
the culture is grown using a selective culture media. By "selective
culture media" we mean a culture media which will support the
specific growth of the organism or organisms of interest while
inhibiting the growth of non-target organisms which might cause
non-specific signal in the assay. For example, Applicants are aware
of certain organisms having endogenous peroxidase activity which
will generate signal, in the presence of the chemiluminescent
substrate used in the assay.
[0068] After the bacteria 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 necessarily, part of the in-situ
hybridization process.
[0069] Using probe-based in-situ analysis of the isolation medium
after organism growth, the number of colony forming units (CFU) of
bacteria which are detected by the organism specific probe, can be
counted or scored (manually or by automated methods) after an
appropriate incubation period. Because the bacteria grow rapidly
and the enzyme-linked PNA probes are suitable for high sensitivity
analysis, typically, the assay can be performed in 1-4 hours.
Because the bacterial 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 bacteria is known and
since only viable organisms grow, the CFU's per unit volume of
sample can be directly determined.
d. Kits:
[0070] In yet another embodiment, this invention is directed to
kits suitable for performing an assay which detects the presence,
absence or number of one or more target organisms in a sample.
Target organisms include members of the bacterial species of E.
coli, Staphylococcus aureus, Pseudomonas aeruginosa, Pseudomonas
cepatia, Pseudomonas fluorescens or organisms of a bacterial genus
including the Salmonella genus, Bacillus genus or Pseudomonas
genus. The general and preferred characteristics of PNA probes
suitable for the detection, identification or quantitation of these
target organisms 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 target organisms in a sample
of interest have been previously described herein.
[0071] 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. Preferred
kits of the invention will be prepared for detecting specific
groups of organisms (e.g. classification within species, genus or
USP organisms). In kits which contain sets of probes, wherein each
of at least two probes of the set are used to detect different
target organisms, the probes of the set are preferably labeled with
one or more independently detectable moieties so that each specific
target organism can be individually detected, identified or
quantitated in a single assay.
Exemplary Assay Formats:
[0072] The probes, probe sets, methods and kits of this invention
are suitable for the detection, identification or quantitation of
bacteria of certain genus or species. In preferred embodiments,
in-situ hybridization is used as the assay format for detecting
identifying or quantitating target organisms. Most preferably,
fluorescence 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, as well
as the Examples of this specification.
[0073] Methods used to experimentally test specific PNA probes in
PNA-FISH assays can be found in Examples 9 and 10 of this
specification. The examples contained herein demonstrate that
labeled PNA probes comprising the probing nucleobase sequences
listed in Table 1 are highly specific for detecting target
organisms even when other organisms listed in the table are present
in the assay. The experimental conditions used in the Examples
yield results within approximately 1-4 hours. The identical
experimental protocol was found to be sensitive, reliable and
generally applicable regardless of the nature or sequence of the
PNA probes used.
[0074] 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 or visualized with a film, camera, microscope (See for
example: Examples 9 and 10 contained herein) 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). Slide scanners and flow cytometers are
particularly useful for rapidly quantitating the number of target
organisms present in a sample of interest.
Exemplary Multiplex PNA-FISH Assays:
[0075] Because the PNA probes of this invention can be labeled with
one or more independently detectable moieties, it is possible to
design PNA probe sets wherein each probe of the set is
independently detectable. 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 such as those used in Examples 9 and 10 of this
specification. Thus, an assay wherein a PNA probe set comprising
three PNA probes, each labeled with one of an independently
detectable derivative of coumarin, fluorescein or rhodamine, could
be used to independently detect, identify or quantitate, in the
same assay, each of three different organisms which might be
present in a sample of interest. 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 two or more organisms in a single sample or assay. By
versatile we mean that the method is generally applicable despite
substantial variability in the nucleobase sequences of the probes
used in the assay.
[0076] The rational described above for performing multiplex assays
can be further extended. For example, any three independently
detectable moieties can be used to detect, identify or quantitate
more than three different organisms of interest provided certain of
the probes of a set can be labeled with two or more independently
detectable moieties. Several alternative methods for labeling PNAs
have been previously referred to herein. Judicious choice of
labeling reagents and labeling methodologies can be used to
introduce multiple independently detectable moieties into a single
PNA probe.
[0077] Table 2 lists probes of an exemplary PNA probe set which
could be used to independently detect 5 species of bacteria and the
Salmonella genus in a single assay. As illustrated in the table,
each of the PNA probes used to detect the Pseudomonas aeruginosa,
Pseudomonas cepatia and Pseudomonas fluorescens species could be
labeled with only one of the three fluorophores whereas the PNA
probes for detection of the E. coli, Staphylococcus aureus species
and bacteria of the Salmonella genus could be labeled with each of
two of the fluorophores wherein the combination of the two
fluorophores is unique to each PNA probe. Using this configuration
of labeled PNA probes, all of the bacteria listed in Table 2 can be
independently detected, identified or quantitated since each
organism of interest will correlate with a unique independently
detectable fluorophore or combination of two independently
detectable fluorophores. For example, Pseudomonas aeruginosa will
exhibit a blue signal whereas E. coli will exhibit both a blue and
green signal. TABLE-US-00002 TABLE 2 Fluorophore Linked To
Independently Target Probe For Detecting Detectable Visible
Organism Target Organism Color(s) P. aeruginosa Coumarin Blue P.
cepatia Fluorescein Green P. fluorescens Rhodamine Red E. Coli
Coumarin and Fluorescein Blue and Green S. aureus Coumarin and
Rhodamine Blue and Red Salmonella Fluorescein and Rhodamine Green
and Red
[0078] Example 10 of this specification demonstrates the
feasibility of multiplex fluorescent in-situ hybridization using
independently detectable PNA probes wherein at least one probe of
the set is labeled with two fluorochrome moieties. In this example,
all four USP bacteria are individually detectable in an assay
format which allows individual cells to be counted. Thus, it has
been demonstrated that a single assay can be used to detect,
identify and quantitate E. coli, Salmonella, Pseudomonas aeruginosa
and Staphylococcus aureus (the USP bacteria) present in a single
sample using three fluorescent dyes. This assay can be performed in
approximately 3 hours and allows for the sensitive detection,
identification and/or quantitation of each of the USP bacteria.
[0079] Those of ordinary skill in the art will recognize that this
method for increasing the number of organisms which can be
independently detected in a single assay (multiplex analysis) is
limited only by the number of independently detectable moieties
available for use in the assay format and the number of
independently detectable moieties which can be linked to the probes
used in the assay. Thus, this invention contemplates assays wherein
tens or even hundreds of organisms of interest can be simultaneous
detected, identified or quantitated.
Immobilization of Probes to a Surface:
[0080] 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 a target organism of interest. 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, 14:2792-2799 (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.
[0081] 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").
[0082] 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.
[0083] 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.
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 a sample which may contain one or more target
organisms. 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 target
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 target organisms of interest. The arrays of this
invention comprise at least one PNA probe (as described herein)
suitable for the detection, identification or quantitation of at
least one target organism (as described herein). Preferred probing
nucleobase sequences for the immobilized PNA probes are listed in
Table 1.
Exemplary Applications for Using the Invention:
[0085] Whether support bound or in solution, the PNA probes, probe
sets, methods and kits of this invention are particularly useful
for the rapid, sensitive and reliable detection of bacteria
(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.
[0086] Whether support bound or in solution, the PNA probes, probe
sets, methods and kits of this invention are also particularly
useful for the rapid, sensitive and reliable detection of bacteria
(pathogens) in 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.
[0087] 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
[0088] This invention is now illustrated by the following examples
which are not intended to be limiting in any way.
Example 1
Synthesis of bis-(2-methoxyethyl)amidyl-diglycolic acid
[0089] 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
Synthesis of
N-[N''-Fmoc-(2''-aminoethyl)]-N-[N,N'-(2-methoxyethyl)amidyl-diglycolyl]g-
lycine ("Fmoc-"E"aeg-OH")
[0090] To 60 mmol of Fmoc-aeg-OH (PE Biosystems, Foster City,
Calif. 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.
[0091] 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.
[0092] 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.
[0093] 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 26.8 g (47
mmol; 78%). An "E" modification of a PNA or polyamide has the
formula: ##STR2##
Example 3
Synthesis of PNAs
[0094] Unless, otherwise stated, PNAs were synthesized using
commercially available reagents and instrumentation obtained from
PE Biosystems, Foster City, Calif. USA. PNAs possessing C-terminal
modifications were prepared by performing the synthesis using
prederivatized synthesis support or by performing the synthesis
using the Fmoc-K(Mtt)-OH (Bachem, Torrance, Calif., USA, P/N
B-2535) or Fmoc-"E"aeg-OH (prepared as described above)
monomers.
Example 4
Preferred Method for Removal of the Fmoc Protecting Group
[0095] 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 the be treated with labeling reagent (See:
Example 6).
Example 5
Preferred Method for Removal of the Mtt Protecting Group from
Lysine (K)
[0096] The resin (still in the synthesis column) was treated with
10 mL of a solution containing 1% trifluoroacetic acid, 5%
triisopropylsilane (TIS) in dichloromethane by passing the solution
through the column over a period of approximately 15 minutes. After
treatment, the synthesis support was washed with DMF. Prior to
treatment with labeling reagent (See: Example 6), the support was
neutralized by treatment with approximately 10 mL of a solution
containing 5% diisopropylethylamine in DMF.
Note: This procedure was only performed on PNA prepared using
Fmoc-PAL-PEG/PS (PE Biosystems, Foster City, Calif. P/N GEN913384).
It was not performed with Fmoc-XAL-PEG/PS (PE Biosystems, Foster
City, Calif. P/N GEN913394).
Example 6
Preferred Method for Amine Labeling of Support Bound PNA with the
NHS Esters of 5(6)carboxyfluorescein (Flu),
6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid (Cou) or
5(and 6)-carboxy-X-rhodamine (Rox)
[0097] The amino protecting group (Fmoc or Mtt) 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.
[0098] The PNA labeled with both Rox and Flu (Flu-Rox-1: Table 3)
was first treated to remove the Fmoc group. The support was then
treated with the Flu-NHS ester. Next the Mtt group was removed and
the support was then treated with the Rox-NHS ester. The PNA was
then cleaved from the synthesis support, deprotected and
purified.
Example 7
General Procedure for Cleavage, Deprotection and Purification
[0099] 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.
[0100] 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. 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.).
[0101] PNA Oligomers Prepared: TABLE-US-00003 TABLE 3 Probe Target
ID Organism PNA Probe Sequence Flu-1 E. coli
Flu-OO-TCA-ATG-AGC-AAA-GGT-NH.sub.2 Flu-2 S. aureus
Flu-OO-GCT-TCT-CGT-CCG-TTC-NH.sub.2 Flu-3 P aeruginosa
Flu-OO-CTG-AAT-CCA-GGA-GCA-NH.sub.2 Flu-4 P aeruginosa
Flu-OO-AAC-TTG-CTG-AAC-CAC-NH.sub.2 Flu-5 P. cepatia
Flu-OO-CCA-TCG-CAT-CTA-ACA-NH.sub.2 Flu-6 P.
Flu-OO-TCT-AGT-CAG-TCA-GTT-NH.sub.2 fluorescens Flu-7 Pseudomonas
Flu-OO-GCT-GGC-CTA-GCC-TTC-NH.sub.2 genus Flu-8 Pseudomonas
Flu-OO-GTC-CTC-CTT-GCG-GTT-NH.sub.2 genus Flu-9 Pseudomonas
Flu-OO-TTC-TCA-TCC-GCT-CGA-NH.sub.2 genus Flu-10 Salmonella
Flu-OO-CCG-ACT-TGA-CAG-ACC-NH.sub.2 genus Flu-11 Salmonella
Flu-OO-CCT-GCC-AGT-TTC-GAA-NH.sub.2 genus Flu-12 Bacillus
Flu-OO-CTT-TGT-TCT-GTC-CAT-NH.sub.2 genus Cou-10 Salmonella
Cou-OO-CCG-ACT-TGA-CAG-ACC-NH.sub.2 genus Rox-2 S. aureus
Rox-OO-GCT-TCT-CGT-CCG-TTC-NH.sub.2 Flu- E. Coli
Flu-OO-TCA-ATG-AGC-AAA-GGT-EE- Rox-1 OK(Rox)-NH.sub.2 All PNA
sequences are written from the amine (N-) terminus to the carboxyl
(C-) terminus. Flu = 5(6)-carboxyfluorescein, Cou =
6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid; Rox =
5(and 6)-carboxyl-X-rhodamine; E is defined above; O =
8-amino-3,6-dioxaoctanoic acid; and K = the amino acid
L-Lysine.
Example 8
Dot Blot
rRNA Preparation:
[0102] Using a Qiagen kit (P/N 75144), total RNA (including app.
80% rRNA) was isolated from the different bacteria which had been
grown in culture. The amount of RNA isolated was determined by
measuring the absorption at 260 nm.
Hybridization to the Membranes:
[0103] Dot blots were made on nylon membranes obtained from
Gibco-BRL (P/N 14830-012). For the RNA of each cultured bacteria, a
dilution row containing 5 spots was made, starting with a
concentration of 16 mM 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, and 0.17 ng total RNA per spot.
Once the spots had air dried, the membrane was UV-crosslinked and
then stored in a plastic bag until used.
[0104] With reference to FIGS. 1, I-V (Species Specific PNA Probes)
and FIG. 2, I-V (Genus Specific PNA Probes), 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. 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.
[0105] All 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.
The prehybridization buffer was then removed from the bags and
fresh hybridization buffer containing the probe(s) of interest
(Flu-1 through Flu-12 as described above), was(were) added to the
bags.
[0106] The hybridization was performed at 50.degree. C. for 1 hour.
The filters were then washed 3 times in TE-7.5 (10 mM Tris-HCl, pH
7.5, 1 mM EDTA) containing 0.2% SDS. The first wash was at room
temperature for 5 minutes. The second and third washes were at
65.degree. C. for 10-15 minutes each.
Visualization of the Membrane:
[0107] 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 with a rinse
solution (10 mM Tris-HCl, pH 9.5; 10 mM NaCl; and 1 mM MgCl.sub.2).
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 MgCl.sub.2) 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.
[0108] With reference to FIGS. 1-1 through 1-V, the specificity of
the species specific PNA probes was examined. PNA probes Flu-1,
Flu-2, Flu-5 and Flu-6 were shown to be specific, within the
parameters of the experiment, for the rRNA of the intended target
organism (E. coli, S. aureus, P. cepatia and P. fluorescens,
respectively) in the presence of rRNA of the other bacteria spotted
on the membrane. The mixture of Flu-3 and Flu-4 was likewise shown
to be specific, within the parameters of the experiment, for the
rRNA of Pseudomonas aeruginosa.
[0109] With reference to FIGS. 2-I through 2-IV, the specificity of
the genus specific PNA probes was examined. PNA probes, Flu-10,
Flu-11 and Flu-12, were shown to be specific, within the parameters
of the experiment, for the rRNA of the intended target organism
(Salmonella genus and Bacillus genus, respectively) in the presence
of rRNA of the other bacteria spotted on the membrane. The mixture
of Flu-7, Flu-8 and Flu-9, was likewise shown to be specific,
within the parameters of the experiment, for the rRNA of the
bacteria of the Pseudomonas genus which were examined.
Example 9
PNA-FISH
[0110] Individual 3 mL cultures of bacteria were grown overnight in
Tryptic Soy Broth (TSB) at 30.degree. C. The broth was then
analyzed for absorbance at 600 nm and then diluted into fresh TSB
until the absorbance at 600 nm was 0.5 OD/mL. These diluted culture
stocks were 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, pelleted again and
resuspended in Fixation Buffer (3% paraformaldehyde in PBS (7 mM
Na.sub.2HPO.sub.4; 3 mM NaH.sub.2PO.sub.4; 130 mM NaCl)). 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
were then used after 30 minutes of incubation or optionally stored
at -20.degree. C. for up to several weeks before being used in an
experiment.
[0111] For each sample prepared, 100 .mu.l of fixed cells in 50%
aqueous ethanol was removed and centrifuged at 8000 R.P.M. for 2
min. The ethanol was then remove from the sample and the pellet was
resuspended in 100 .mu.l of sterile PBS and pelleted again by
centrifugation at 8000 R.P.M. for 2 min.
[0112] 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 (e.g. Flu-1 thought Flu-12) at a concentration of 30 pmol/mL.
The hybridization was performed at 55.degree. C. for 30
minutes.
[0113] The sample was then centrifuged at 8000 R.P.M. for 2 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 5 minutes. The
sample was then centrifuged at 8000 rpm for 5 minutes. The TE-9.0
was then removed from the pellet. The TE-9.0 wash was then repeated
two more times.
[0114] After the final wash the cells were resuspended in 100 .mu.l
PBS. 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. A coverslip was added to the slide and its
position fixed using a couple of drops of nail polish. Each of the
nine different bacterial strains, Pseudomonas fluorescens,
Pseudomonas aeruginosa, Pseudomonas cepatia, Pseudomonas putida,
Escherichia coli, Bacillus subtilis, Staphylococcus epidermidis,
Staphylococcus aureus, and Salmonella typhimurium, were fixed and
hybridized with each of probes Flu-1 through Flu-6 following the
protocol described above. After hybridization and wash, each strain
was spotted and mounted on a microscope slide (see above) and
examined using a Nikon fluorescent microscope equipped with a
60.times. immersion oil objective, a 10.times. ocular (total
enlargement is 600 fold), and an Omega Optical XF22 filter. All
probes exhibited an excellent discrimination between the bacteria
strain which they were selected to detect, and the other strains
tested. Even the detection of Pseudomonas fluorescens using probe
Flu-6 showed excellent specificity, despite the low level of cross
hybridization observed in the dot blots (see FIG. 1-V). Similarly,
hybridization of each of the nine strains with the genus specific
probes (# Flu-10 though Flu-12) or probe mixtures (# Flu-7 through
Flu-9) showed excellent specificity for the individual genus
tested.
Example 10
Multiplex PNA-FISH
[0115] A mixture of the four USP organisms, E. coli, S. aureus, P.
aeruginosa, and S. typhimurium, fixed individually as previously
described, was prepared and hybridized with a mixture of four PNA
probes comprised of Flu-Rox-1, Rox-2, Flu-4 and Cou-10 (See Table
3). The hybridization protocol was as described in Example 9.
[0116] After hybridization and wash, the bacteria were spotted and
mounted on a microscope slide (see above) and inspected 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 XF05 (blue) filter). Electronic digital images were made
of the slide using a SPOT CCD-camera and software obtained from
Diagnostic Instruments, Inc., Sterling Heights, Mich. (USA).
[0117] The digital images obtained, all covering the same section
of the slide, are presented in FIG. 3-I through 3-IV. In FIG. 3-I
(green image), bacteria stained green by the Flu-3 (P. aeruginosa)
and Flu-Rox-1 (E. coli) probes are visible. In FIG. 3-II (red
image), bacteria stained red by the Flu-Rox-1 (E. coli) and the
Rox-2 (S. aureus) probes are visible. In FIG. 3-III (blue image),
bacteria stained blue by the Cou-10 probe (S. typhimurium) are
visible. In FIG. 3-IV, a digital composite of the blue and the red
images is presented. This digital composite clearly demonstrates
that simultaneous detection of several different bacteria 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.
Equivalents
[0118] 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
12 1 15 DNA Artificial Sequence Description of Combined DNA/RNA
MoleculePNA Probing Nucleobase Sequence 1 tcaatgagca aaggt 15 2 15
DNA Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 2 gcttctcgtc cgttc 15 3 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 3 ctgaatccag gagca 15 4 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 4 aacttgctga accac 15 5 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 5 ccatcgcatc taaca 15 6 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 6 tctagtcagt cagtt 15 7 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 7 gctggcctag ccttc 15 8 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 8 gtcctccttg cggtt 15 9 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 9 ttctcatccg ctcga 15 10 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 10 ccgacttgac agacc 15 11 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 11 cctgccagtt tcgaa 15 12 15 DNA
Artificial Sequence Description of Combined DNA/RNA MoleculePNA
Probing Nucleobase Sequence 12 ctttgttctg tccat 15
* * * * *