Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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MONITORING HIGH-RISK ENVIRONMENTS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to continuation-in-part of U.S. Serial No.
10/306,113, filed November 27, 2002 and U.S. Application No. 101392,041 filed
March
18, 2003.
TECHNICAL FIELD
This invention relates to monitoring high-risk environments for microbes,
including microbial pathogens, and more particularly to detecting microbes in
high-risk
environments by detecting microbial markers.
BACKGROUND
From a public health perspective, the detection of microbes, particularly
microbial
pathogens, in environments such as food processing facilities and health care
institutions,
is critically important. For example, nosocomial outbreaks, or infectious
outbreaks that
occur in excess of generally 2 times the normal expectancy in patients in
health care
15 institutions, present difficult issues of associated morbidity, mortality,
and expense in a
health care system already crippled by rising insurance and health care costs.
The CDC
has reported that nearly $5 billion are added to U.S. health costs every year
as a result of
nosocomial infections. Similarly, according to the CDC's National Nosocomial
Surveillance System, the rate of hospital-related fungal infections nearly
doubled between
20 1980 and 1990. In 1997, an estimated 240,000 individuals showed clinical
symptoms of
endemic mycoses, with the mortality rate in patients with systemic fungal
infections
ranging from 30-100%, depending on the pathogen. Over-reliance on antibiotics
has led
to antibiotic resistant bacterial strains, themselves often of nosocomial
origin. Finally,
pathogen contamination in environments such as food processing plants and day-
care
25 centers has led to widespread infectious outbreaks, often with intensive
and expensive
investigation required on the local or national level to determine and control
the source of
the pathogen. Thus, knowledge of the nature and amount of a microbial pathogen
in a
particular environment may be required for efficient infectious disease source
identification, outbreak management, and treatment.
3o Microbial profiles are representations of individual strains, subspecies,
species,
andlor genera of microorganisms within a community of microorganisms.
Generally,
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determining a microbial profile involves taxonomic and/or phylogenetic
identification of
the microbes in a community. A microbial profile also can include quantitative
information about one or more members of the community. Once one or more
microorganisms have been identified in a microbial community, microbial
profiles can be
presented as, for example, lists of microorganisms, graphical or tabular
representations of
the presence and/or numbers of microorganisms, or any other appropriate
representation
of the diversity and/or population levels of the microorganisms in a
community.
Microbial profiles are useful for identifying pathogenic and non-pathogenic
microbial
organisms in biological and non-biological samples (e.g., samples from
animals, the
environment, or inanimate obj ects).
A microbial profile can be determined using any of a number of known methods.
For example, the microbes in a sample can be cultured and colonies identified
and/or
enumerated. It has been estimated, however, that culturing typically recovers
only about
0.1 % of the microbial species in a sample (based on comparisons between
direct
15 microscopic counts and recovered colony-forming units). Culture-independent
methods
to determine microbial profiles can include extracting and analyzing microbial
macromolecules from a sample. Useful target molecules typically include those
that as a
class are found in all microorganisms, but are diverse in their structures and
thereby
reflect the diversity of the microbes. For example, various nucleic acid-based
assays can
2o be employed to determine a microbial profile. Some nucleic acid-based
population
methods use denaturation and reannealing kinetics to derive an indirect
estimate of the
guanine and cytosine (%G+C) content of the DNA in a sample, for example. The
%G+C
technique provides an overall view of the microbial community, but typically
is sensitive
only to massive changes in the make-up of the community
25 Genetic fingerprinting is another nucleic-acid based method that can be
used to
determine a microbial profile. Genetic fingerprinting utilizes random-sequence
oligonucleotide primers that hybridize specifically to random sequences
throughout the
genome. Amplification results in a multitude of products, and the distribution
of those
products is referred to as a genetic fingerprint. Particular patterns can be
associated with
3o a community of microbes in the sample. Genetic fingerprinting, however,
lacks the
ability to conclusively identify specific microbial species.
Denaturing or temperature gradient gel electrophoresis (DGGE or TGGE) is
another nucleic acid-based technique that can be used to determine a microbial
profile.
.As amplification products are electrophoresed in gradients with increasing
denaturant or
2
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temperature, the double-stranded molecule melts and its mobility is reduced.
The melting
behavior is determined by the nucleotide sequence, and unique sequences will
resolve
into individual bands. Thus, a D/TGGE gel yields a genetic fingerprint
characteristic of
the microbial community, and the relative intensity of each band reflects the
abundance of
the corresponding microorganism. An alternative format includes single-
stranded
conformation polymorphism (SSCP). SSCP relies on the same physical basis as
%G+C
renaturation methods, but reflects a significant improvement over such
methods.
In addition, a microbial profile can be determined using terminal restriction
fragment length polymorphism (TRFLP). Amplification products can be analyzed
for the
presence of known sequence motifs using restriction endonucleases that
recognize and
cleave double-stranded nucleic acids at these motifs. Alternative approaches
include
"amplified ribosomal DNA restriction analysis (AADRA)" in which the entire
amplification product, rather than just the terminal fragment, is considered.
A microbial profile also can be determined by cloning and sequencing microbial
~ 5 nucleic acids present in a biological or non-biological sample (e.g., a
biological sample
from an animal). Cloning of individual nucleic acids into Esclae~ichia coli
and
sequencing each nucleic acid gives the highest density of information but
requires the
most effort. Although sequencing nucleic acids is automated, routine
monitoring of
changes in the microbial profile of an animal by cloning and sequencing
nucleic acids
2o from the microorganisms still requires considerable time and effort.
Therefore, despite the existence of methods for determining microbial
profiles,
there remains a need for rapid, sensitive, and quantitative methods capable of
detecting
and identifying microbes, particularly microbial pathogens, in high-risk
environments.
25 SUMMARY
The invention is based on the discovery that the presence or absence of a
microbe
in a high-risk environment can be determined quickly and sensitively by
detecting the
presence and/or concentration of a microbial marker, specifically a cpn60
marker, in a
sample obtained from the high-risk envirorunent. Chaperonin 60 (cpn60) markers
are
3o particularly useful for determining the presence of a microbe in a sample
and optionally
determining a microbial profile of a sample. Chaperonin proteins are molecular
chaperones required for proper folding of polypeptides in vivo. cpn60 is found
universally in prokaryotes and in the organelles of eukaryotes, and can be
used as a
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species-specific target and/or probe for identification and classification of
microorganisms. Sequence diversity within this protein-encoding gene appears
greater
between and within bacterial genera than for 16S rDNA sequences, thus making
cpn60 a
superior target sequence having more distinguishing power for microbial
identification at
the species level than 16S rDNA sequences.
Accordingly, the detection of the presence and/or concentration of the cpn60
marker may be capable of providing a microbial profile of the sample. In
particular,
microbial profiles of biological and non-biological samples from a high-risk
environment
can be determined using methods that involve detection of cpn60 markers,
including
cpfz60-specific nucleic acid molecules and cpn60-specific polypeptides.
Methods of the invention are rapid and sensitive, and can be used to detect
the
presence or absence of cpn60-containing microbes in general, as well as to
identify what
species of microbes are present and in what amounts. Using cpf260 primers,
probes, and
antibodies, methods of the invention can include amplifying cpn60-specific
nucleic acids
15 and detecting amplification products using techniques such as fluorescence
resonance
energy transfer (FRET). Other rapid and sensitive methods for detecting and
quantifying
cph60-specific nucleic acids include fluorescent in situ hybridization (FISH),
for
example. Accordingly, primers and probes for detecting cprZ60-containing
microbial
species axe provided by the invention, as are methods for using such primers
and probes
2o and kits containing such primers and probes. Similarly, the invention also
provides
methods for detecting cpn60-specific polypeptides, such as enzyme-linked
immunoassays
(ELISAs), or other polypeptide detection methods, including surface plasmon
resonance
techniques, mass spectrometry, and electrophoretic methods. Accordingly, kits
containing
cpn60-specific antibodies are also contemplated by the present invention.
25 In one aspect, the invention provides methods for monitoring a high-risk
environment for the presence or absence of one or more microbes. Such a method
includes providing a sample obtained from the high-risk environment; and
detecting the
presence or absence of a cpn60 marker in the sample. Generally, the presence
of the
cpra60 marker is indicative of the presence of the one or more microbes.
3o In one embodiment, the detecting step is capable of providing a microbial
profile
of the sample. Typically, the microbial profile includes identifying one or
more microbes
in the sample, and may further include quantifying the amount of one or more
microbes in
the sample. In addition, a microbial profile of the high-risk environment can
be acquired
and compared at two or more points, for example, time points or location
points. Further,
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a control microbial profile from a control sample can be acquired from the
high-risk
environment, which can be compared to the sample microbial profile.
Generally, the cpn60 marker is a cprt60-specific nucleic acid or a cpn60-
specific
polypeptide. In one embodiment, the cpzz60-specific nucleic acid is a genomic
nucleic
acid coding sequence of a cpn60 protein, for example, of chromosomal origin.
In another
embodiment, the cpn60-specific nucleic acid is an amplified sequence of a
cpn60 coding
sequence of the microbe.
Generally, the detecting step can be a nucleic acid-based assay or a
polypeptide-
based assay. Representative nucleic acid-based assays include PCR and FISH
assays,
while representative polypeptide-based assays include an immunodiagnostic
assay (e.g.,
ELISA), a mass spectrometric technique, and a surface plasmon resonance
technique.
Typically, a microbe that can be detected by methods of the invention include
bacteria, protozoa, rickettsiae, and fungi. Representative bacterial microbes
include the
Staphylococcus, Streptococcus, Pseudomonas, Escherichia, Bacillus, Brucella,
15 Clzlamydia, Clostridium, Shigella, Mycobacterium, Agrobacterium,
Bartonella, Borellia,
Bradyrhizobiutn, Ehrlichia, Haetnophilus, Helicobacter, Heliobacter,
Lactobacillus,
Neisseria, Rhizobium, Streptomyces, Synechocoecus, ~ymotnottas,
Syneclzocyotis,
Mycoplastna, Yersinia, Tlibrio, Burkholderia, Fratzciscella, Legionella,
Sahnoztella,
Bifzdobacteriutn, Enterococcus, Enterobacter, Citrobacter, Bacteroides,
Prevotella,
2o Xanthomonas, ~ylella, and Campylobacter genera. Representative protozoan
microbes
include Acanthamoeba, Cryptosporidium, and Tetrahymena genera. Representative
fungal microbes include Aspergillus, Colletrotrichunz, Cochliobolus,
Helminthosporium,
Microcyclus, Puccinia, Pyricularia, I~euterophoma, Monilia, Candida, and
Sacclzarottzyces. Representative rickettsiae microbes include Coxiella
burnetti,
25 Bartonella quintana, Rochlimea Quintatta, Rickettsia Quintana, Rickettsia
prowasecki,
and Rickettsia rickettsii.
Examples of high-risk environments include a retail food industry facility, a
school, a medical environment, a water facility, a residence, a food transport
vehicle, a
processing facility, or a research facility. For example, a retail food
industry facility can
3o be a butcher shop, a grocery store, a restaurant, a cafeteria, an
entertainment facility (e.g.,
a theater, a park, a zoo, a rink, an arena, a civic center, a museum, or a
stadium), or a
convenience store; a medical environment can be a hospital, a physician's
office, a dental
office, a clinic, a nursing home, an outpatient facility, a physical therapy
facility, a spa, an
operating room, or a medical diagnostic laboratory; a water facility can be a
wastewater
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treatment plant, a potable water facility, a desalinization facility, a
recycled water facility,
an aquaculture facility, an air conditioning unit, a humidifier, a water
storage tank, a
water fountain, a fire hydrant, a tub, a hot tub, a sauna, a steam bath, or a
water tap; a
food transport vehicle can be a truck, a rail car, or a ship; and a processing
facility can be
a food processing facility (e.g., an abbatoir, a packaging facility, a
purification plant, or a
fermentation vessel), a chemical processing facility, or a biological
processing facility.
In one embodiment, the sample is a tissue sample. A tissue sample can be a
biopsy sample, or can be derived from a swab of an animal. Representative
animals
include a human, a cow, a pig, a horse, a goat, a sheep, a dog, a cat, a bird,
a monkey, a
1 o fish; a clam, an oyster, a mussel, a lobster, a shrimp, and a crab.
Representative tissue
samples from such animals include an eye, a tongue, a cheek, a hoof, a beak, a
snout, a
foot, a hand, a mouth, a teat, the gastrointestinal tract, a feather, an ear,
a nose, a mucous
membrane, a scale, a shell, the fur, and the skin.
In another embodiment, the sample is a fomite or is derived from a fomite
present
~ 5 in the high-risk environment.
In another embodiment, the sample is a food sample such as a prepared food
sample, a raw food sample, a cooked food sample, or a perishable food sample.
Representative food samples include beef, pork, poultry, seafood, dairy (milk,
eggs, or
cheese), fruit, vegetable, seed, nut, and fungus. Further, the sample can be a
liquid
2o sample such as a water sample, a blood sample, a urine sample, a lachrymal
sample, a
sweat sample, a saliva sample, a lymph sample, and a cerebrospinal fluid
sample.
In another aspect, the invention provides an article of manufacture. An
article of
manufacture of the invention includes at least one cpn60 antibody, wherein the
cpn60
antibody is attached to a solid support; and an indicator molecule. An article
of
25 manufacture also can include instructions for using the cpn60 antibody to
detect a cpn60-
containing microbe. A representative solid support is a dipstick.
Unless otherwise defined, all technical and scientific terms used herein have
the
same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. Although methods and materials similar or equivalent to
those
3o described herein can be used in the practice or testing of the present
invention, suitable
methods and materials are described below. In addition, the materials,
methods, and
examples are illustrative only and not intended to be limiting. All
publications, patent
applications, patents, and other references mentioned herein are incorporated
by reference
6
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WO 2004/051226 PCT/US2003/038814
in their entirety. In case of conflict, the present specification, including
definitions, will
control.
The details of one or more embodiments of the invention are set forth in the
accompanying drawings and the description below. Other features, objects, and
advantages of the invention will be apparent from the drawings and detailed
description,
and from the claims.
DESCRIPTION OF DRAWINGS
FIG 1 is the sequence of a cpn60 gene from Clostridiurra perfringens (SEQ ID
NO:1; GenBank° Accession No. NC 003366). Sequences to which the
universal cpn60
primers described herein can hybridize (or the complement thereof) are
underlined.
FIG 2 is the sequence of a cpn60 gene from EschericlZia coli (SEQ ID N0:2;
GenBank~ Accession No. NC 000913). Sequences to which the universal cpn60
primers
described herein can hybridize (or the complement thereof) are underlined.
FIG. 3 is the sequence of a cpn60 gene from Staphyloc~ccus coelicolor (SEQ ID
~5 N0:3; GenBank~ Accession No. AL939121). Sequences to which the universal
cpn60
primers described herein can hybridize (or the complement thereof) are
underlined.
' FIG. 4 is the sequence of a cpn60 gene from CampylobacteY jejur2i (SEQ ID
N0:4; GenBank~ Accession No. NC 002163). Sequences to which the universal
cpra60
primers described herein can hybridize (or the complement thereof) are
underlined.
2o FIG 5 is the sequence of a cpra60 gene from Salmonella enterica (SEQ ID
NO:S;
GenBank~ Accession No. NC 003198). Sequences to which the universal cpn60
primers
described herein can hybridize (or the complement thereof) are underlined.
Like reference symbols in the various drawings indicate like elements.
DETAILED DESCRIPTION
25 The present invention provides methods for monitoring high-risk
environments
-for the presence or absence of one or more microbes. The microbes may be
pathogens.
In particular, the presence or absence of a microbe in a high-risk environment
can be
determined quickly and sensitively by detecting the presence and/or
concentration of a
microbial marker, specifically a cpn60 marker, in a sample obtained from the
high-risk
30 environment.
High-Risk Environments
7
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As used herein, a high-risk environment is an environment at risk of
contamination by one or more microbes because of the nature of the activity
conducted
therein or because of the environment's potential to be a source of a microbe.
High-risk
environments include, but are not limited to, the following: a retail food
industry facility,
a school, a medical environment, a water facility, a residence, perishable
foods or
improperly preserved foods, a transportation facility, a processing facility,
and a research
facility. For example, the retail food industry has traditionally been a
source of serious
infectious outbreaks, and is an example of one type of high-risk environment.
Retail food
industry locations includes such places as a butcher shop, a grocery store, a
restaurant, a
1 o cafeteria, an entertainment facility, and a convenience store.
Entertainment facilities include such places as theaters, libraries, malls,
parks,
zoos, rinks, arenas, civic centers, museums, amusement parks, arcades,
athletic fields and
locations, conference halls, meeting rooms, and stadiums. Entertainment
facilities may
be high-risk environments because, for example, food is processed, prepared,
and sold on-
~ 5 site and because of the likelihood of inoculating a large number of humans
in such
locations.
Medical environments are also examples of high-risk environments. Medical
environments generally have a close physical association of numerous patients
with a
variety of illnesses, many of who are already in an immunocompromised state.
The
20 potential for cross-contamination, nosocomial outbreaks, and the
development of
antibiotic resistant strains is high in such an environment. Nonlimiting
examples of
medical environments include a hospital, a physician's office, a dental
office, a clinic, a
nursing home, an outpatient facility, a physical therapy facility, a spa, an
operating room,
and a medical diagnostic laboratory.
25 Water facilities are yet additional examples of high-risk environments. For
example, wastewater treatment plants are naturally confronted with a variety
of pathogens
in the water to be treated. Aquaculture facilities, such as fish farms, oyster
beds, etc., are
also susceptible to infectious outbreaks. Air conditioning units have faced
increased
scrutiny as a source of infectious agents after the Legionnaire's outbreak.
Hot tubs have
3o been recently implicated as a source of Mycobacterium aviurn infections
("hot tub lung")
in people who use them frequently. Other examples of water facilities include
potable
water facilities, desalinization facilities, dams, recycled water facilities,
humidifiers,
water storage tanks, potable water reservoirs (e.g., water coolers), water
fountains, fire
hydrants, tubs, saunas, steam baths, and water taps.
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Transportation facilities are additional examples of high-risk environments. A
transportation facility may be high-risk because it is used, e.g., to
transport food. For
example, food transport vehicles such as railway cars, trucks, tank cars, and
shipping
vessels that transport bulk quantities of food may need to be monitored prior
to loading
and after off loading. Alternatively, a transportation facility may be a high-
risk
enviromnent because of the potential for a microbe to be carned through such a
facility
Non-limiting examples include a car, a bus, a plane, a train, a bicycle, a
motorcycle, a
ship, an airport, a bus ten~ninal, a train terminal, a port, a Custom's
checkpoint, and an
immigration checkpoint. For example, contaminated food (e.g., fruit) may be
carried
1 o through an immigration checkpoint.
Processing facilities, including food chemical, and biological processing
facilities, are other examples of high-risk environments. Food processing
facilities have
been under increasing pressure to control microbial contamination of processed
foods,
such as by requiring the implementation of Hazard Analysis and Critical
Control Point
~ 5 Plans (HACCP) and antimicrobial intervention techniques. Food processing
facilities
include abbatoirs (slaughter-houses), packaging facilities, purification (e.g.
radiation,
pasteurization, fumigation) facilities, storage locations (e.g., .silos,
vessels, tanks), and
fen~nentation vessels. Chemical and biological processing facilities can
include analytical
laboratories, production plants, pilot plants, and purification plants.
2o Foods, particularly perishable foods and improperly preserved, stored, or
handled
foods, are also examples of high-risk environments. Perishable foods include,
for
example, milk, eggs, cheeses, breads, buffet table menu items, carry-out menu
items,
vegetables, and fruits. Improperly preserved foods include those that are
commercially
preserved (e.g., canned, sealed, jarred, bagged etc. by a commercial food
source) or self
25 preserved (e.g., home canning, etc.). The food may be prepared, e.g., in a
restaurant or a
home kitchen. Such a prepared food sample may be either cooked or raw (e.g.,
salads,
juices, fruits). Alternatively, the food may be unprocessed. Typical food
products include
beef, pork, poultry, seafood, dairy, fruit, vegetable, seed, nut, fungus, and
grain. Dairy
food samples include milk, eggs, butter, and cheese, as well as condiments and
sauces
3o prepared from such dairy foods (e.g., mayonnaise, aioli, cream sauces,
hollandaise sauces,
etc.).
Sample types and sampling methods
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The methods described herein are capable of detecting the presence or absence
of
a microbe, and optionally a microbial profile, based on the presence of a
cpra60 marker in
a sample obtained from a high-risk environment. Microbial profiles can be
determined
for biological or non-biological samples.
As used herein, "biological sample" refers to any sample obtained, directly or
indirectly, from a subject animal or control animal. Representative biological
samples
that can be obtained from an animal include or are derived from biological
tissues,
biological fluids, and biological elimination products (e.g., feces).
Biological tissues can
include biopsy samples or swabs of the biological tissue of interest, e.g.,
nasal swabs,
o throat swabs, dermal swabs. The tissue can be any appropriate tissue from an
animal,
such as a human, cow, pig, horse, goat, sheep, dog, cat, bird, monkey, fish,
clam, oyster,
mussel, lobster, shrimp, and crab. Depending on the microbe and the type of
high-risk
environment, the tissue of interest to sample (e.g., by biopsy or swab) can be
an eye, a
tongue, a cheek, a hoof, a beak, a snout, a foot, a hand, a mouth, a teat, the
15 gastrointestinal tract, a feather, an ear, a nose, a mucous membrane, a
scale, a shell, the
fur, and the skin.
Biological fluids can include bodily fluids (e.g., urine, milk, lachrymal
fluid,
vitreous fluid, sputum, cerebrospinal fluid, sweat, lymph, saliva, semen,
blood, or serum
or plasma derived from blood); a lavage such as a breast duct lavage, lung
lavage, a
2o gastric lavage, a rectal or colonic lavage, or a vaginal lavage; an
aspirate such as a nipple
or teat aspirate; a fluid such as a cell culture or a supernatant from a cell
culture; and a
fluid such as a buffer that has been used to obtain or resuspend a sample,
e.g., to wash or
to wet a swab in a swab sampling procedure. Biological samples can be obtained
from an
animal using methods and techniques known in the art. See, for example,
Diagfaostic
25 Molecular Microbiology: Prifzciples and Applications (Persing et al. (eds),
1993,
American Society for Microbiology, Washington D.C).
Biological samples also can be obtained from the environment (e.g., air,
water, or
soil). Methods are known for extracting biological samples (e.g., cells) from
such
environments. Additionally, a biological sample suitable for use in the
methods of the
3o invention can be a substance that one or more animals have contacted. For
example, an
aqueous sample from a water bath, a chill tank, a scald tank, or other aqueous
environments with which a subject or control animal has been in contact, can
be used in
the methods of the invention to evaluate a microbial profile. A soil sample
that one or
more subject or control animals have contacted, or on which an animal has
deposited
l0
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WO 2004/051226 PCT/US2003/038814
fecal or other biological material, also can be used in the methods of the
invention. For
example, nucleic acids can be isolated from such biological samples using
methods and
techniques known in the art. See, for example, Diagnostic Molecular
Microbiology:
Principles and Applications (Persing et al. (eds), 1993, American Society for
Microbiology, Washington D.C).
The methods of the present invention can also be used to detect the presence
of
microbes and/or microbial pathogens in or on non-biological samples. For
example, a
fomite present in a high-risk environment may be sampled to detect the
presence or
absence of a microbe. A fomite is a physical (inanimate) object that serves to
transmit, or
o is capable of transmitting, an infectious agent, e.g., a microbial pathogen,
from animal to
animal. (It is noted that inanimate objects such as food, air, and liquids are
not considered
fomites, but are considered infectious "vehicles," or media that are
'routinely taken into
the body.) Indeed, one study that evaluated the presence of Salfnonella spp.,
Listeria spp.,
and Yersinia spp. pathogenic microbes on various abbatoir fomites detected
Salrnoraella
~ 5 spp. on 11.1 % of meat cleavers, 6.25 % of worktables, and 5.6% of floors;
Yersinia
enterocolitica was found on 16.7% of slaughter floors and on 12.5% of
worktables; and
L isteria naonocytogenes was isolated from 13.3% of cold room floor swabs and
on 7.1
of hand-wash basins. See Kathryn Cooper, Guelph Food Technology Centre, "The
Plant
Environment Counts: Protect your Product through Environmental Sampling," Meat
&
2o Poultry, May 1999. Nonlimiting examples of fomites include utensils,
knives, drinking
glasses, food processing equipment, cutting surfaces, cutting boards, floors,
ceilings,
walls, drains, overhead lines, ventilation systems, waste traps, troughs,
machines, toys,
storage boxes, toilet seats, door handles, clothes, gloves, bedding, combs,
shoes, changing
tables (e.g., for diapers), diaper bins, toy bins, food preparation tables,
food transportation
25 vehicles (e.g., rail cars and shipping vessels), gates, ramps, floor mats,
foot pedals of
vehicles, sinks, washing facilities, showers, tubs, buffet tables, surgical
equipment and
instruments, and analytical instruments and equipment.
A microbe may be left as a residue on a fomite. In such cases, it is important
to
detect accurately the presence of the pathogen on the fomite in order to
prevent the spread
so of the pathogen. For example, it is known that microbes may exist in viable
but
noncuhturable forms on fomites, or that nonculturable bacteria of selected
species can be
resuscitated to a culturable state under certain conditions. Often such
nonculturable
bacteria are present in biofilins on fomites. Accordingly, detection methods
that rely on
cuhturable forms may significantly under-report microbial contamination on
fomites. The
11
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WO 2004/051226 PCT/US2003/038814
methods of the present invention, including PCR-based methods, can aid in the
detection
of microbes, particularly nonculturable forms, by amplification and detection
of cpn60-
specific nucleic acid sequences.
The sample from the high-risk environment can also be a food sample. For
example, the sample may be a prepared food sample, e.g., from a restaurant.
Such a
prepared food sample may be either cooked or raw (e.g., salads, juices,
fruits). In other
embodiments, the food sample may be unprocessed and/or raw, e.g., a tissue
sample of an
animal from a slaughterhouse, either prior to or after slaughter. The food
sample may be
perishable. Typically food samples will be taken from food products such as
beef, pork,
1 o poultry, seafood, dairy, fruit, vegetable, seed, nut, fungus, and grain.
Dairy food samples
include milk, eggs, butter, and cheese, as well as sauces and condiments made
from the
same.
Methods for collecting and storing biological and non-biological samples are
generally known to those of skill in the art. For example, the Association of
Analytical
~ 5 Communities International (AOAC International) publishes and validates
sampling
techniques for testing foods and agricultural products for microbial
contamination. See
also WO 9832020 (PCT/WO 97US04289) and US Pat. No..5,624,810, which set forth
methods and devices for collecting and concentrating microbes from the air,
liquid, or a
surface. WO 9832020 also provides methods for removing somatic cells, or
animal body
2o cells present at varying levels in certain samples.
In particular embodiments of the methods described herein, a separation and/or
concentration step may be necessary to separate any microbes present from
other
components of a sample or to concentrate the microbe to an amount sufficient
for rapid
detection. For example, a sample suspected of containing a biological microbe
may
25 require a selective enrichment of the microbe (e.g., by culturing in
appropriate media,
e.g., for 4-96 hours, or longer) prior to employing the detection methods
described herein.
Alternatively, appropriate filters and/or immunomagnetic separations can
concentrate a
microbe without the need for an extended growth stage. For example, antibodies
specific
for a cpn60-specific polypeptide can be attached to magnetic beads and/or
particles.
3o Multiplexed separations, in which two or more concentration processes are
employed, are
also contemplated, e.g., centrifugation, membrane filtration, electrophoresis,
ion
exchange, affinity chromatography, and immunomagnetic separations.
Certain air or water samples may need to be concentrated. For example, certain
air sampling methods require the passage of a prescribed volume of air over a
filter to trap
12
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
any microbes, followed by isolation into a buffer or liquid culture.
Alternatively, the
focused air is passed over a plate (e.g., agar) medium for growth of any
microbe.
Methods for sampling a tissue or a fomite with a swab axe known to those of
skill
in the art. Generally, a swab is hydrated (e.g., with an appropriate buffer,
such as Cary-
Blair medium, Stuart's medium, Amie's medium, PBS, buffered glycerol saline,
or water)
and used to sample an appropriate surface (a fomite or tissue) for a microbe.
Any
microbe present is then recovered from the swab, such as by centrifugation of
the
hydrating fluid away from the swab, removal of supernatant, and resuspension
of the
centrifugate in an appropriate buffer, or by washing of the swab with
additional diluent or
buffer. The so-recovered sample may then be analyzed according to the methods
described herein for the presence of a microbe. Alternatively, the swab may be
used to
culture a liquid or plate (e.g., agar) medium in order to promote the growth
of any
microbes for later testing. Suitable swabs include both cotton and sponge
swabs; see, for
example, those provided by Tecra~, such as the Tecra ENVIROSWAB~.
~5 The samples from the high-risk environment case be used "as is," or may
need to
be treated prior to application of the detection methods employed herein. ~'or
example,
samples can be processed (e.g., by nucleic acid or protein extraction methods
andlor kits
known in the art) to release nucleic acid or proteins. In other cases, a
biological sample
can be contacted directly with PCR reaction components and appropriate
oligonucleotide
2o primers and probes.
Detection of cpn60 markers
Methods provided herein are useful for determining the presence of one or more
microbes and/or microbial pathogens in a high-risk environment and optionally
provide
25 microbial profiles of the high-risk environment. As used herein, "microbes"
refers to
bacteria, protozoa, rickettsiae, and fungi. Microbial communities for which a
microbial
profile can be generated can include but are not limited to the following
examples of
prokaryotic genera: Staphylococcus, Streptococcus, Pseudonaonas, Escherichia,
Bacillus,
Brucella, Chlamydia, Clostridium, Shigella, Mycobacterium, Agrobacterium,
Bartonella,
3o Borellia, Bradyrhizobiurra, Ehrlichia, Haemophilus, Helicobacter,
Heliobacter
Lactobacillus, Neisseria, Rhizobium, Streptomyces, Synecl2ococcus, Zymomonas,
Synechocyotis, Mycoplasma, Yersinia, Vibrio, Bufkholderia, Franciscella,
Legionella,
Salmonella, Bifidobacter-ium, Enterococcus, Enterobacter, Citrobacter,
Bacteroides,
Prevotella, Xantlzomonas, Xylella, and Campylobacter; the following examples
of
13
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
protozoa genera: Acanthamoeba, CYyptosporidium, and Tetrahymena; the following
examples of fungal genera: AspeYgillus, ColletYOtrichum, Cochliobolus,
Helmintlaosporium, Micnocyclus, Puccinia, Pyricularia, Deuterophonaa, Monilia,
Candida, and Saccharomyces; and the following rickettsiae microbes: Coxiella
burnetti,
Bar~tonella quintana, Rochlimea Quintana, Rickettsia Quintana, Rickettsia
prowasecki,
and Rickettsia rickettsii.
The detection of a microbe or microbial profile in a sample (e.g., a
biological
sample or a non-biological sample) obtained from a high-risk environment can
be
determined using methods that involve detection of a cpn60 marker. cpn60
markers
include cpn60-specific nucleic acids and cpn60-specific polypeptides. As used
herein, a
cpn60-specific nucleic acid is a nucleic acid that includes, is complementary
to, or
specifically hybridizes to all or a portion of the genomic cpn60 nucleic acid
sequence.
Typically, cpn60-specific nucleic acids are defined with reference to exons,
although
introns and regulatory sequences associated with cpra60 coding sequences are
also within
~5 the scope of the present invention. The term "nucleic acid" as used herein
encompasses
both RNA and DNA, including genomic DNA. The nucleic acid can be double-
stranded
or single-stranded. The nucleic acid can contain one or more restriction
sites.
Generally, a cpn60-specific nucleic acid marker will be all or a portion of
the
genomic nucleic acid coding sequence of a cpra60 protein. A cpn60-specific
nucleic acid
2o may be specific to a particular species of microbe or may be universal.
Species-specific
cpn60-specific nucleic acid sequences are cpra60 nucleic acid sequences that
hybridize
preferentially to cpn60 nucleic acid sequences from a given species under
appropriate
assay conditions. One of skill in the art can design probes to detect such
species-specific
cpn60-specific nucleic acid sequences by e.g., aligning cpn60 nucleic acid
coding
25 sequences and looking for variable regions, e.g., sequences that would not
cross-hybridize
under the appropriate assay conditions to cpn60 nucleic acid sequences from
other
species. Alternatively, one of skill in the art will recognize that variable
regions, e.g.,
those that demonstrate no more than 99% sequence similarity (e.g., no more
than 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and 98% sequence
3o similarity) to a cpn60-specific nucleic acid from another species may also
be useful as
species-specific cpn60-specific nucleic acids. Use of such specific probes in
the methods
described herein allows the discriminatory detection of a particular species
in a sample.
In calculating percent sequence identity, two sequences are aligned and the
number of identical matches of nucleotides or amino acid residues between the
two
14
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
sequences is determined. The number of identical matches is divided by the
length of the
aligned region (i. e., the number of aligned nucleotides or amino acid
residues) and
multiplied by 100 to arnve at a percent sequence identity value. It will be
appreciated
that the length of the aligned region can be a portion of one or both
sequences up to the
full-length size of the shortest sequence. It will be appreciated that a
single sequence can
align differently with other sequences and hence, can have different percent
sequence
identity values over each aligned region. It is noted that the percent
identity value is
usually rounded to the nearest integer. For example, 78.1 %, 78.2%, 78.3%, and
78.4%
are rounded down to 78%, while 78.5%, 78.6%, 78.7%, 78.8%, and 78.9% are
rounded
1 o up to 79%. It is also noted that the length of the aligned region is
always an integer.
The alignment of two or more sequences to determine percent sequence identity
is
performed using the algorithm described by Altschul et al. (1997, Nucleic
Acids Res.,
25:3389-3402) as incorporated into BLAST (basic local alignment search tool)
programs,
available at http://www.ncbi.nlm.nih.~ov. BLAST searches can be performed to
~5 determine percent sequence identity between a apn60-specific nucleic acid
sequence from
one organism and a cpn60-specific nucleic acid sequence from another organism
aligned
using the Altschul et al. algorithm. BLASTN is the program used to align and
compare
the identity between nucleic acid sequences, while BLASTP is the program used
to align
and compare the identity between amino acid sequences. When utilizing BLAST
2o programs to calculate the percent identity between cpn60 sequences, the
default
parameters of the respective programs are used.
As used herein, a "universal" cpn60-specific nucleic acid is a cpn60 nucleic
acid
sequence that is capable of hybridizing under the appropriate assay conditions
to one or
more cpn60 nucleic acid coding sequences from other microbes. Such sequences,
of
25 course, would not hybridize to non-cpn60 nucleic acids under the same assay
conditions.
One of skill in the axt will recognize that hybridization assay conditions can
be
manipulated in a variety of ways to increase or decrease stringency, e.g., by
salt,
temperature, choice of buffer, etc. See e.g., Sambrook et al., Molecular
Cloning; A
LabonatoYy Manual, 2na Ed., Cold Spring Harbor Laboratory Press, 1989.
Alternatively,
30 one of skill in the art will recognize that nucleic acid sequences
demonstrating greater
than 75%, 80%, 85%, 90%, or 95% sequence similarity to at least a second cpn60
nucleic
acid sequence may be useful as universal cpn60-specific nucleic acids. For
example,
cpn60 coding sequences from a particular bacterial genera (e.g.,
Staphylococcus), or
sequences derived therefrom, may cross-hybridize under the appropriate assay
conditions
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
or have sufficiently similar sequences to one or more cpn60 coding sequences
within
members of the genus or across genera. As one of skill in the art will
recognize, and as
explained more fully below, a "universal" probe can then be designed that is
capable of
detecting two or more of such similar sequences in a sample. For example, one
of skill in
the art can align cpn60 coding sequences (e.g., from a given genera) and look
for
sequences that have sequence identity; these sequences thus would be capable
of cross-
hybridizing to two or more members of the genera. In addition, varying
hybridization
stringencies can be tested to ascertain optimal conditions for cross-
hybridization.
Detection of such universal cpn60-specific nucleic acids allows the detection
of two or
to more microbes in a sample, e.g., the detection of all members of a genera,
as described
previously.
As used herein, a cpn60-specific polypeptide marker is a polypeptide that
includes
all or a portion of a cpn60 protein: As with cpf260-specific nucleic acids, a
cpn60-specific
polypeptide marker can be specific to a particular microbial species or
universal. A
~ 5 species-specific cpn60-specific polypeptide marker is all or a portion of
a given species'
cpn60 protein. In the methods of the present invention, the probe or
analytical method for
detecting the marker should be capable of discriminating between the
particular cpn60-
specific polypeptide and all other cpn60-specific polypeptides, e.g., by mass
in mass-
spectrometry applications or by a particular epitope in an antibody assay. For
example,
2o and as described more fully below, one of skill in the art will recognize
that antibodies,
particularly monoclonal antibodies, can be obtained that recognize an epitope
that is
specific to a particular species' cpn60 protein. Accordingly, use of such
specific
antibodies in the methods described herein allows the differential detection
of a particular
species in a sample.
25 In other embodiments, a cpn60-specific polypeptide marker can be universal.
For
example, a "universal" cpn60-specific polypeptide marker may be a common
structural
(conformational) epitope in two or more cpn60 proteins. As described more
fully below,
antibodies, particularly polyclonal antibodies, raised against cpn60 proteins
or
polypeptides may be screened for cross-reactivity to common epitopes on cpn60-
specific
3o polypeptides from two or more microbes.
16
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WO 2004/051226 PCT/US2003/038814
Nucleic acid-based assays
Real-tune PCR assays
Nucleic acid-based methods for identifying and/or quantitating the amount of a
microbe in a sample can include amplification of a cpn60 nucleic acid.
Amplification
methods such as PCR provide powerful means by which to increase the amount of
a
particular nucleic acid sequence. Nucleic acid hybridization also can be
included in
determining the presence or absence of a microbe in a sample. Probing
amplification
products with species-specific hybridization probes is one of the most
powerful analytical
tools available for profiling. The physical matrix for hybridization can be a
nylon
o membrane (e.g., a macroarray) or a microarray (e.g., a microchip),
incorporation of one or
more hybridization probes into an amplification reaction (e.g., TaqMan~ or
Molecular
Beacon technology), solution-based methods (e.g., ORIGEN technology), or any
one of
numerous approaches devised for clinical diagnostics. As discussed above,
probes can be
designed to preferentially hybridize to amplification products from individual
species or
to discriminate specific species.
U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose
conventional PCR techniques. PCR typically employs t<vo oligonucleotide
primers that
bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in
the
present invention include oligonucleotide primers capable of acting as a point
of initiation
of nucleic acid synthesis within or adjacent to cpn60 sequences (see below). A
primer
can be purified from a restriction digest by conventional methods, or can be
produced
synthetically. Primers typically are single-stranded for maximum efficiency in
amplification, but a primer can be double-stranded. Double-stranded primers
are first
denatured (e.g., treated with heat) to separate the strands before use in
amplification.
Primers can be designed to amplify a nucleotide sequence from a particular
microbial
species, or can be designed to amplify a sequence from more than one species.
Primers
that can be used to amplify a nucleotide sequence from more than one species
are referred
to herein as "universal primers."
PCR assays can employ template nucleic acids such as DNA or RNA, including
3o messenger RNA (mRNA). The template nucleic acid need not be purified; it
can be a
minor fraction of a complex mixture, such as a microbial nucleic acid
contained in animal
cells. Template DNA or RNA can be extracted from a biological or non-
biological
sample using routine techniques such as those described in Diagnostic
Molecular
Microbiology: Principles arid Applicatioras (Parsing et. al. (ads.), 1993,
American Society
17
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
for Microbiology, Washington D.C.). Nucleic acids can be obtained from any of
a
number of sources, including plasmids, bacteria, yeast, organelles, and higher
organisms
such as plants and animals. Standard conditions for generating a PCR product
are well
known in the art (see, e.g., PCR Pri~aeY: A Laboratory Manual, Dieffenbach and
Dveksler (eds.), Cold Spring Harbor Laboratory Press, 1995).
Once a PCR amplification product is generated, it can be detected by, for
example, hybridization using FRET technology. FRET technology (see, for
example,
U.S. Patent Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on
the concept
that when a donor fluorescent moiety and a corresponding acceptor fluorescent
moiety are
positioned within a certain distance of each other, energy transfer taking
place between
the two fluorescent moieties can be visualized or otherwise detected and
quantitated.
Two oligonucleotide probes, each containing a fluorescent moiety, can
hybridize to an
amplification product at particular positions determined by the
complementarity of the
oligonucleotide probes to the target nucleic acid sequence. Upon hybridization
of the
oligonucleotide probes to the amplification product at the appropriate
positions, a FRET
signal is generated. Hybridization temperatures and times.can range from about
35°C to
about 65°C for about 10 seconds to about 1 minute. Detection of FRET
can occur in real-
time, such that the increase in an amplification product after each cycle of a
PCR assay is
detected and, in some embodiments, quantified.
2o Fluorescent analysis and quantification can be carried out using, for
example, a
photon counting epifluorescent microscope system (containing the appropriate
dichroic
mirror and filters for monitoring fluorescent emission in a particular range
of
wavelengths), a photon counting photomultiplier system, or a fluorometer.
Excitation to
initiate energy transfer can be earned out with an argon ion laser, a high
intensity mercury
arc lamp, a fiber optic light source, or another high intensity light source
appropriately
filtered for excitation in the desired range.
Fluorescent moieties can be, for example, a donor moiety and a corresponding
acceptor moiety. As used herein with respect to donor and corresponding
acceptor
fluorescent moieties, "corresponding" refers to an acceptor fluorescent moiety
having an
3o emission spectrum that overlaps the excitation spectrum of the donor
fluorescent moiety.
The wavelength maximum of the emission spectrum of an acceptor fluorescent
moiety
typically should be at least 100 nm greater than the wavelength maximum of the
excitation spectrum of the donor fluorescent moiety, such that efficient non-
radiative
energy transfer can be produced therebetween.
1s
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
Fluorescent donor and corresponding acceptor moieties are generally chosen for
(a) high efficiency Forster energy transfer; (b) a large final Stokes shift
(>100 nm); (c)
shift of the emission as far as possible into the red portion of the visible
spectrum (>600
nm); and (d) shift of the emission to a higher wavelength than the Raman water
fluorescent emission produced by excitation at the donor excitation
wavelength. For
example, a donor fluorescent moiety can be chosen with an excitation maximum
near a
laser line (for example, Helium-Cadmium 442 nm or Argon 488 nm), a high
extinction
coefficient, a high quantum yield, and a good overlap of its fluorescent
emission with the
excitation spectrum of the corresponding acceptor fluorescent moiety. A
corresponding
1o acceptor fluorescent moiety can be chosen that has a high extinction
coefficient, a high
quantum yield, a good overlap of its excitation with the emission of the donor
fluorescent
moiety, and emission in the red part of the visible spectrum (>600 nm).
Representative donor fluorescent moieties that can be used with various
acceptor
fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, B-
~5 phycoerythrin, 9-acridineisothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-
isothio-
cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-
isothiocyanatophenyl)-4-
methylcoumarin, succinimdyl 1-pyrenebutyrate, and 4-acetamido-4'-
isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative
acceptor
fluorescent moieties, depending upon the donor fluorescent moiety used,
include LCTM_
2o Red 640, LCTM-Red 705, CyS, Cy5.5, Lissamine rhodamine B sulfonyl chloride,
tetramethyl rhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine
isothiocyanate, fluorescein, diethylenetriamine pentaacetate, and other
chelates of
Lanthanide ions (e.g., Europium, or Terbium). Donor and acceptor fluorescent
moieties
can be obtained from, for example, Molecular Probes, Inc. (Eugene, OR) or
Sigma
25 Chemical Co. (St. Louis, MO).
Donor and acceptor fluorescent moieties can be attached to probe
oligonucleotides
via linker arms. The length of each linker arm is important, as the linker
arms will affect
the distance between the donor and acceptor fluorescent moieties. The length
of a linker
arm for the purpose of the present invention is the distance in Angstroms (A)
from the
3o nucleotide base to the fluorescent moiety. In general, a linker arm is from
about 10 to
about 25 A in length. The linker arm may be of the kind described in WO
84/03285, for
example. WO 84/03285 also discloses methods for attaching linker arms to a
particular
nucleotide base, as well as methods for attaching fluorescent moieties to a
linker arm.
19
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
An acceptor fluorescent moiety such as an LCTM-Red 640-NHS-ester can be
combined with C6-Phosphoramidites (available from ABI (Foster City, CA) or
Glen
Research (Sterling, VA)) to produce, for example, LCTM-Red 640-
Phosphoramidite.
Linkers frequently used to couple a donor fluorescent moiety such as
fluorescein to an
oligonucleotide include thiourea linkers (FITC-derived, for example,
fluorescein-CPG's
from Glen Research or ChemGene (Ashland, MA)), amide-linkers (fluorescein-NHS-
ester-derived, such as fluorescein-CPG from BioGenex (San Ramon, CA)), or 3'-
amino-
CPG's that require coupling of a fluorescein-NHS-ester after oligonucleotide
synthesis.
Using commercially available real-time PCR instrumentation (e.g.,
LightCyclerTM,
o Roche Molecular Biochemicals, Indianapolis, Il~, PCR amplification,
detection, and
quantification of an amplification product can be combined in a single closed
cuvette with
dramatically reduced cycling time. Since detection and quantification occur
concurrently
with amplification, real-time PCR methods obviate the need for manipulation of
the
amplification product, and diminish the risk of cross-contamination between
amplification products. Real-time PCR greatly reduces turn-around time and is
an
attractive alternative to conventional PCR techniques in the clinical
laboratory, in the
Field, or at the point of care.
Conventional PCR methods in conjunction with FRET technology can be ased to
practice the methods of the invention. In one embodiment, a LightCyclerTM
instrument is
2o used. A detailed description of the LightCyclerTM System and real-time and
on-line
monitoring of PCR can be found at the Roche website. The following patent
applications
describe real-time PCR as used in the LightCyclerTM technology: WO 97/46707,
WO
97/46714, and WO 97/46712. The LightCyclerTM instrument is a rapid thermal
cycler
combined with a microvolume fluorometer utilizing high quality optics. This
rapid
thermocycling technique uses thin glass cuvettes as reaction vessels. Heating
and cooling
of the reaction chamber are controlled by alternating heated and ambient air.
Due to the
low mass of air and the high ratio of surface area to volume of the cuvettes,
very rapid
temperature exchange rates can be achieved within the LightCyclerTM thermal
chamber.
Addition of selected fluorescent dyes to the reaction components allows the
PCR to be
3o monitored in real-time and on-line. Furthermore, the cuvettes serve as an
optical element
for signal collection (similar to glass fiber optics), concentrating the
signal at the tip of the
cuvette. The effect is efficient illumination and fluorescent monitoring of
microvolume
samples.
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
The LightCyclerTM carousel that houses the cuvettes can be removed from the
instrument. Therefore, samples can be loaded outside of the instrument (in a
PCR Clean
Room, for example). In addition, this feature allows for the sample carousel
to be easily
cleaned and sterilized. The fluorometer, as part of the LightCyclerTM
apparatus, houses
the light source. The emitted light is filtered and focused by an epi-
illumination lens onto
the top of the cuvette. Fluorescent light emitted from the sample is then
focused by the
same lens, passed through a dichroic mirror, filtered appropriately, and
focused onto data-
collecting photohybrids. The optical unit currently available in the
LightCyclerTM
instrument (Roche Molecular Biochemicals, Catalog No. 2 O11 46~) includes
three band-
o pass filters (530 nm, 640 nm, and 710 nm), providing three-color detection
and several
fluorescence acquisition options. Data collection options include once per
cycling step
monitoring, fully continuous single-sample acquisition for melting curve
analysis,
continuous sampling (in which sampling frequency is dependent on sample
number)
and/or stepwise measurement of all samples after defined temperature interval.
~ 5 The LightCyclerTM can be operated and the data retrieved using a PC
workstation
and a Windows operating system. Signals from the samples are obtained as the
machine
positions the capillaries sequentially over the optical unit. The software can
display the
presence and amount of fluorescent signals in real-time irmnediately after
each
measurement. Fluorescent acquisition time is 10-100 milliseconds (msec). After
each
20 cycling step, a quantitative display of fluorescence vs. cycle number can
be continually
updated for all samples. The generated data can be stored for further
analysis.
Real-time PCR methods include multiple cycling steps, each step including an
amplification step and a hybridization step. In addition, each cycling step
typically is
followed by a FRET detecting step to detect hybridization of one or more
probes to an
25 amplification product. The presence of an amplification product is
indicative of the
presence of one or more cph60-containing species. As used herein, "cpn60-
containing
species" refers to microbes that contain cpn60 nucleic acid sequences.
Generally, the
presence of h'RET indicates the presence of one or more cpn60-containing
species in the
sample, and the absence of FRET indicates the absence of a cpra60-containing
species in
3o the sample. Typically, detection of FRET within, for example, 20, 25, 30,
35, 40, or 45
cycling steps is indicative of the presence of a cpn60-containing species.
As described herein, cph60 amplification products can be detected using
labeled
hybridization probes that take advantage of FRET technology. A common format
of
FRET technology utilizes two hybridization probes that generally are designed
to
21
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
hybridize in close proximity to each other, where one probe is labeled with a
donor
fluorescent moiety and the other is labeled with a corresponding acceptor
fluorescent
moiety. Thus, two cpn60 probes can be used, one labeled with a donor
fluorophore and
the other labeled with a corresponding acceptor fluorophore. The presence of
FRET
s between the donor fluorescent moiety of the first cpn60 probe and the
corresponding
acceptor fluorescent moiety of the second cpya60 probe is detected upon
hybridization of
the cph60 probes to the cpra60 amplification product. For example, a donor
fluorescent
moiety such as fluorescein is excited at 470 nm by the light source of the
LightCyclerTM
Instrument. During FRET, the fluorescein transfers its energy to an acceptor
fluorescent
1 o moiety such as LightCyclerTM-Red 640 (LCTM-Red 640) or LightCyclerTM-Red
705
(LCTM-Red 705). The acceptor fluorescent moiety then emits light of a longer
wavelength, which is detected by the optical detection system of the
LightCyclerTM
instrument. Efficient FRET can only take place when the fluorescent moieties
are in
direct local proximity and when the emission spectrum of the donor fluorescent
moiety
~5 overlaps with the absorption spectrum of the acceptor fluorescent moiety.
The intensity
of the emitted signal can be correlated with the number of original target DNA
molecules
(e.g., the number of copies of cpra60). .
Another FRET format can include the use of TaqMan~' technology to detect the
presence or absence of a cpn60 amplification product, and hence, the presence
or absence
20 of cpn60-containing species. TaqMan~ technology utilizes one single-
stranded
hybridization probe labeled with two fluorescent moieties. When a first
fluorescent
moiety is excited with light of a suitable wavelength, the absorbed energy is
transferred to
a second fluorescent moiety according to the principles of FRET. The second
fluorescent
moiety generally is a quencher molecule. During the annealing step of the PCR
reaction,
25 the labeled hybridization probe binds to the target DNA (i.e., the cpya60
amplification
product) and is degraded by the 5' to 3' exonuclease activity of the Taq
Polymerase
during the subsequent elongation phase. As a result, the excited fluorescent
moiety and
the quencher moiety become spatially separated from one another. As a
consequence of
excitation of the first fluorescent moiety in the absence of the quencher, the
fluorescence
3o emission from the first fluorescent moiety can be detected. By way of
example, an ABI
PRISM~ 7700 Sequence Detection System (Applied Biosystems, Foster City, CA)
uses
TaqMari technology, and is suitable for performing the methods described
herein for
detecting cpn60-containing species. Information on PCR amplification and
detection
22
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
using an ABI PRISM~ 770 system can be found at the Applied Biosystems website
(world wide web at appliedbiosystems.comlproducts).
Molecular beacons in conjunction with FRET also can be used to detect the
presence of a cpn60 amplification product using the real-time PCR methods of
the
invention. Molecular beacon technology uses a hybridization probe labeled with
a first
fluorescent moiety and a second fluorescent moiety. The second fluorescent
moiety
generally is a quencher, and the fluorescent labels typically are located at
each end of the
probe. Molecular beacon technology uses an oligonucleotide probe having
sequences that
permit secondary structure formation (e.g., a hairpin). As a result of
secondary structure
1 o formation within the probe, both fluorescent moieties are in spatial
proximity when the
probe is in solution. After hybridization to the target cpn60 amplification
product, the
secondary structure of the probe is disrupted and the fluorescent moieties
become
separated from one another such that after excitation with light of a suitable
wavelength,
the emission of the first fluorescent moiety can be detected.
~ 5 The amount of FRET corresponds to the amount of amplification product,
which
in turn corresponds to the amount of template nucleic acid present in the
sample.
Similarly, the amount of template nucleic acid corresponds to the amount of
microbial
organism present in the sample. Therefore, the amount of FRET produced when
amplifying nucleic acid obtained from a biological sample can be correlated to
the
2o amount of a microorganism. Typically, the amount of a microorganism in a
sample can
be quantified by comparing to the amount of FRET produced from amplified
nucleic acid
obtained from known amounts of the microorganism (e.g., a standard curve).
Accurate
quantitation requires measuring the amount of FRET while amplification is
increasing
linearly. In addition, there must be an excess of probe in the reaction.
Furthermore, the
25 amount of FRET produced in the known samples used for comparison purposes
can be
standardized for particular reaction conditions, such that it is not necessary
to isolate and
amplify samples from every microorganism for comparison purposes.
As an alternative to FRET, a cpn60 amplification product can be detected
using,
for example, a fluorescent DNA binding dye (e.g., SYBRGreenI~ or SYBRGoId~
30 (Molecular Probes)). Upon interaction with an amplification product, such
DNA binding
dyes emit a fluorescent signal after excitation with light at a suitable
wavelength. A
double-stranded DNA binding dye such as a nucleic acid intercalating dye also
can b,e
used. When double-stranded DNA binding dyes are used, a melting curve analysis
usually is performed for confirmation of the presence of the amplification
product.
23
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
Melting curve analysis is an additional step that can be included in a cycling
profile. Melting curve analysis is based on the fact that a nucleic acid
sequence melts at a
characteristic temperature (Tm), which is defined as the temperature at which
half of the
DNA duplexes have separated into single strands. The melting temperature of a
DNA
molecule depends primarily upon its nucleotide composition. A DNA molecule
rich in G
and C nucleotides has a higher Tm than one having an abundance of A and T
nucleotides.
The temperature at which the FRET signal is lost correlates with the melting
temperature
of a probe from an amplification product. Similarly, the temperature at which
signal is
generated correlates with the annealing temperature of a probe with an
amplification
o product. The melting temperatures) of cpn60 probes from an amplification
product can
confirm the presence or absence of cpn60-containing species in a sample, and
can be used
to quantify the amount o.f a particular cpn60-containing species. For example,
a universal
probe that hybridizes to a variable region within cpn60 will have a Tm that
depends upon
the sequence to which it hybridizes. Thus, a universal probe may have a Tm of
70°C
when hybridized to a cpn60 amplification product generated from one microbial
organism, but a Tm of 65°C when hybridized to a cpn60 amplification
product generated
from a second microbial organism. By observing a temperature-dependent, step-
wise
decrease in fluorescence of a sample as it is heated; the particular cpn60-
containing
species in the sample can be identified and the relative amounts of the
species in the
2o sample can be determined.
Within each thermocycler run, control samples can be cycled as well. Positive
control samples can amplify a nucleic acid control template (e.g., a nucleic
acid other
than cpn60) using, for example, control primers and control probes. Positive
control
samples also can amplify, for example, a plasmid construct containing a cpra60
nucleic
acid molecule. Such a plasmid control can be amplified internally (e.g.,
within the
sample) or in a separate sample run side-by-side with the test samples. Each
thermocycler run also should include a negative control that, for example,
lacks cpn60
template DNA. Such controls are indicators of the success or failure of the
amplification,
hybridization and/or FRET reaction. Therefore, control reactions can readily
determine,
3o for example, the ability of primers to anneal with sequence-specificity and
to initiate
elongation, as well as the ability of probes to hybridize with sequence-
specificity and for
FRET to occur.
In one embodiment, methods of the invention include w:eps to avoid
contamination. For example, an enzymatic method utilizing uracil-DNA
glycosylase is
24
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
described in U.S. Patent Nos. 5,035,996, 5,683,896 and 5,945,313, and can be
used to
reduce or eliminate contamination between one thermocycler run and the next.
In
addition, standard laboratory containment practices and procedures are
desirable when
performing methods of the invention. Containment practices and procedures
include, but
are not limited to, separate work areas for different steps of a method,
containment hoods,
barrier filter pipette tips and dedicated air displacement pipettes.
Consistent containment
practices and procedures by personnel are necessary for accuracy in a
diagnostic
laboratory handling clinical samples.
It is understood that the present invention is not limited by the
configuration of
one or more commercially available instruments.
Fluorescent it2 situ hybridization (FISH)
In situ hybridization methods such as FISH also can be used to determine a
microbial profile. In general, in situ hybridization methods provided herein
include the .
steps of fixing a biological sample, hybridizing a cpn60 probe to target DNA
contained
within the fixed biological sample, washing to remove non-specific binding,
detecting the
hybridized probe, and quantifying the amount of hybridized probe.
Typically, cells are harvested from a biological sample using standard
techniques.
For example, cells can be harvested by centrifuging a biological sample and
resuspending
2o the pelleted cells in, for example, phosphate-buffered saline (PBS). After
re-centrifuging
the cell suspension to obtain a cell pellet, the cells can be fixed in a
solution such as an
acid alcohol solution, an acid acetone solution, or an aldehyde such as
formaldehyde,
paraformaldehyde, or glutaraldehyde. For example, a fixative containing
methanol and
glacial acetic acid in a 3:1 ratio, respectively, can be used as a fixative. A
neutral
buffered formalin solution also can be used (e.g., a solution containing
approximately 1
to 10% of 37-40% formaldehyde in an aqueous solution of sodium phosphate).
Slides
containing the cells can be prepared by removing a majority of the fixative,
leaving the
concentrated cells suspended in only a portion of the solution.
The cell suspension is applied to slides such that the cells do not overlap on
the
3o slide. Cell density can be measured by a light or phase contrast
microscope. For
example, cells harvested from a 20 to 100 ml urine sample typically are
resuspended in a
final volume of about 100 to 200 Tl of fixative. Three volumes of this
suspension (e.g., 3,
10, and 30 Tl), are then dropped into 6 mm wells of a slide. The cellularity
(i.e., the
density of cells) in these wells is then assessed with a phase contrast
microscope. If the
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
well containing the greatest volume of cell suspension does not have enough
cells, the
cell suspension can be concentrated and placed in another well.
Probes for FISH are chosen for maximal sensitivity and specificity. Using a
set of
probes (e.g., two or more cpn60 probes) can provide greater sensitivity and
specificity
than the use of any one probe. Probes typically are about 50 to about 2 x 103
nucleotides
in length (e.g., 50, 75, 100, 200, 300, 400, 500, 750, 1000, 1500, or 2000
nucleotides in
length). Longer probes cal comprise smaller fragments of about 100 to about
500
nucleotides in length. Probes that hybridize with locus-specific DNA can be
obtained
commercially from, for example, Vysis, Inc. (Downers Grove, IL), Molecular
Probes,
Inc. (Eugene, OR), or from Cytocell (Oxfordshire, UK). Alternatively, probes
can be
made non-commercially from chromosomal or genomic DNA through standard
techniques. For example, sources of DNA that can be used include genomic DNA,
cloned DNA sequences, somatic cell hybrids that contain one, or a part of one,
human
chromosome along with the normal chromosome complement of the host, and
~5 chromosomes purified by flow cytometry or microdissection. The region of
interest can
be isolated through cloning, or by site-specific amplification.via PCR. See,
for example,
Nath and Johnson, Biotechraic Histochem., 1998, 73(1):6-22, Wheeless et al.,
Cytometyy,
1994, 17:319-326, and U.S. Patent No. 5,491,224.
Probes for FISH typically are directly labeled with a fluorescent moiety (also
2o referred to as a fluorophore), an organic molecule that fluoresces after
absorbing light of
lower wavelength/higher energy. The fluorescent moiety allows the probe to be
visualized without a secondary detection molecule. After covalently attaching
a
fluorophore to a nucleotide, the nucleotide can be directly incorporated into
a probe using
standard techniques such as nick translation, random priming, and PCR
labeling.
25 Alternatively, deoxycytidine nucleotides within a probe can be
transaminated with a
linker. A fluorophore then can be covalently attached to the transaminated
deoxycytidine
nucleotides. See, U.S. Patent No. 5,491,224. The amount of fluorophore
incorporated
into a probe can be known or determined, and this value in turn can be used to
determine
the amount of nucleic acid to which the probe binds. In conjunction with
analysis of
3o samples (e.g., a serial dilution of a sample) containing known numbers of
microbial
organisms, the number of microbial organisms in a biological or non-biological
sample
can be determined.
When more than one probe is used, fluorescent moieties of different colors can
be
chosen such that each probe in the set can be distinctly visualized and
quantitated. For
26
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
example, a combination of the following fluorophores may be used: 7-amino-4-
methylcoumarin-3-acetic acid (AMCA), Texas Reds (Molecular Probes, Inc.), 5-
(and-
6)-carboxy-X-rhodamine, lissamine rhodamine B, 5-(and-6)-carboxyfluorescein,
fluorescein-5-isothiocyanate (FITC), 7-diethylaminocoumarin-3-carboxylic acid,
tetramethylrhodamine-5-(and-6)-isothiocyanate, 5-(and-6)-
carboxytetramethylrhodamine,
7-hydroxycoumarin-3-carboxylic acid, 6-[fluorescein 5-(and-6)-
carboxamido]hexanoic
acid, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionic
acid, eosin-5-
isothiocyanate, erythrosin-5-isothiocyanate, and CascadeTM blue acetylazide
(Molecular
Probes, Inc.). Probes can be viewed with a fluorescence microscope and an
appropriate
o filter for each fluorophore, or by using dual or triple band-pass filter
sets to observe
multiple fluorophores. See, for example, U.S. Patent No. 5,776,688.
Alternatively,
techniques such as flow cytometry can be used to examine and quantitate the
hybridization pattern of the probes.
Probes also can be indirectly labeled with biotin or digoxygenin, or labeled
with
~5 radioactive isotopes such as 32P and 3H, although secondary detection
molecules or
further processing then may be required to visualize the probes and quantify
the amount
of hybridization. For example, a probe indirectly labeled with biotin can be
detected and
quantitated using avidin conjugated to a detectable enzymatic marker such as
alkaline
phosphatase or horseradish peroxidase. Enzymatic markers can be detected and
2o quantitated in standard colorimetric reactions using a substrate and/or a
catalyst for the
enzyme. Catalysts for alkaline phosphatase include 5-bromo-4-chloro-3-
indolylphosphate and nitro blue tetrazolium. Diaminobenzoate can be used as a
catalyst
for horseradish peroxidase.
Prior to in situ hybridization, the probes and the chromosomal DNA contained
2s within the cell each are denatured. Denaturation typically is performed by
incubating in
the presence of high pH, heat (e.g., temperatures from about 70°C to
about 95°C), organic
solvents such as formamide and tetraalkylammonium halides, or combinations
thereof.
For example, chromosomal DNA can be denatured by a combination of temperatures
above 70°C (e.g., about 73°C) and a denaturation buffer
containing 70% formamide and
30 2.X SSC (0.3 M sodium chloride and 0.03 M sodium citrate). Denaturation
conditions
typically are established such that cell morphology is preserved. Probes can
be denatured
by heat (e.g., by heating to about 73°C for about five minutes).
After removal of denaturing chemicals or conditions, probes are annealed to
the
chromosomal. DNA under hybridizing conditions. "Hybridizing conditions" are
27
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
conditions that facilitate annealing between a probe and target chromosomal
DNA.
Hybridization conditions vary, depending on the concentrations, base
compositions,
complexities, and lengths of the probes, as well as salt concentrations,
temperatures, and
length of incubation. The higher the concentration of probe, the higher the
probability of
forming a hybrid. For example, in situ hybridizations typically are performed
in
hybridization buffer containing 1-2X SSC, 50% formaxnide, and blocking DNA to
suppress non-specific hybridization. In general, hybridization conditions, as
described
above, include temperatures of about 25°C to about 55°C, and
incubation times of about
0.5 hours to about 96 hours. More particularly, hybridization can be performed
at about
32°C to about 40°C for about 2 to about 16 hours.
Non-specific binding of probes to DNA outside of the target region can be
removed by a series of washes. The temperature and concentration of salt in
each wash
depend on the desired stringency. For example, for high stringency conditions,
washes
can be carned out at about 65°C to about 80°C, using 0.2X to
about 2X SSC, and about
0.1 % to about 1 % of a non-ionic detergent such as Nonidet P-40 (NP40).
Stringency can
be lowered by decreasing the temperature of the washes or by increasing the
concentration of salt in the washes.
f~aRNA-based assays
2o Alternatively, in order to test for the presence or absence of, or measure
the level
of, a cpn60-specific mRNA in a sample, e.g., a sample comprising cells, the
cells can be
lysed and total RNA can be purified or semi-purified from lysates by any of a
variety of
methods known in the art. Methods of detecting or measuring levels of
particular mRNA
transcripts are also familiar to those in the art. Such assays include,
without limitation,
hybridization assays using detectably labeled cpn60-specific nucleic acid (DNA
or RNA)
probes and quantitative or semi-quantitative RT-PCR methodologies employing
appropriate cpn60 oligonucleotide primers. Additional methods for quantitating
mRNA
in cell lysates include RNA protection assays and serial analysis of gene
expression
(SAGE). Alternatively, qualitative, quantitative, or semi-quantitative in situ
hybridization
3o assays can be carried out using, for example, samples such as tissue
sections or unlysed
cell suspensions, and detectably (e.g., fluorescently, isotopically, or
enzymatically)
labeled DNA or RNA probes.
Polypeptide-Based Assays
28
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
The invention also features polypeptide-based assays. A cpn60 protein, or
cpn60-
specific polypeptide, can be used as a universal target to determine the
presence or
absence of one or more microbes, and further used as species-specific targets
and/or
probes for the identification and classification of specific microbes. Such
assays can be
used on their own or in conjunction with other procedures (e.g., nucleic acid-
based
assays) to monitor high-risk environments.
In the assays of the invention, the presence or absence of a cpn60-specific
polypeptide is detected and/or its level is measured. The presence of a cpn60-
specific
polypeptide may be measured in a liquid sample such as a body fluid (e.g.,
urine, milk,
lachrymal fluid, vitreous fluid, sputum, cerebrospinal fluid, sweat, lymph,
saliva, semen,
blood, or serum or plasma derived from blood); a lavage such as a breast duct
lavage,
lung lavage, a gastric lavage, a rectal or colonic lavage, or a vaginal
lavage; an aspirate
such as a nipple or teat aspirate; a fluid such as a cell culture or a
supernatant from a cell
culture; a fluid such as a buffer that has been used to obtain a sample from
e.g., a fomite,
such as a buffer used to wash or to wet a swab in a swab sampling procedure;
and a water
sample. In addition, any sample that can be solubilized may also be used in
the methods
of the present invention.
Methods of detecting or measuring the levels of a protein of interest (e.g., a
cpn60
protein, or cpn60-specific polypeptides) in cells are known in the art. Many
such
2o methods employ antibodies (e.g., polyclonal antibodies or mAbs) that bind
specifically to
the protein.
Antibodies and antibody-based assays
Antibodies having specific binding affinities for a cpn60 protein or a cpn60-
specific polypeptide may be produced through standard methods. As used herein,
the
terms "antibody" or "antibodies" include intact molecules as well as fragments
thereof
which are capable of binding to an epitopic determinant of a cpn60-specific
polypeptide.
The term "epitope" refers to an antigenic determinant on an antigen to which
the paratope
of an antibody binds. Epitopic determinants usually consist of chemically
active surface
3o groupings of molecules such as amino acids or sugar side chains, and
typically have
specific three-dimensional structural characteristics, as well as specific
charge
characteristics. Epitopes generally have at least five contiguous amino acids
(a
continuous epitope), or alternatively can be a set of noncontiguous amino
acids that
define a particular structure (e.g., a conformational epitope). The terms
"antibody" and
29
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
"antibodies" include polyclonal antibodies, monoclonal antibodies, humanized
or
chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and
F(ab)Z
fragments.
Antibodies may be specific for a particular cpn60-specific polypeptide, e.g.,
the
cpn60 protein of the Clostr~idiurn perfrifagens microbe. Alternatively, they
may be cross-
reactive with two or more cpn60-specific polypeptides, e.g., cross-react or
bind to two or
more cpn60 proteins. For example, such antibodies may bind to common epitopes
present in two or more cpn60 proteins or cpn60-specific polypeptides. As used
herein,
such antibodies with specificity for two or more cpn60-specific polypeptides
are termed
o "universal" antibodies. For example, certain antibodies may bind to common
epitopes
present in all cpn60-specific polypeptides. Certain of such antibodies thus
may be termed
able to detect the presence or absence of any microbe in a sample.
In certain embodiments of the method described herein, depending on the high-
risk environment and the purpose for monitoring, it may be sufficient to
determine simply
~ 5 whether or not any microbe is present, and optionally the relative
concentration or amount
of the microbe. Such a detection may occur through, e.g., the use of one or
more
"universal" cpn60 antibodies, such as an antibody that binds or demonstrates
specificity
to two or more cpn60-specific polypeptides (e.g., one that is cross-reactive
with all cpn60
proteins of a particular genera, or with all bacterial cpn60 proteins) as
described
2o previously.
In other embodiments, the identification of the particular microbe may be
preferred. Accordingly, an antibody specific for a particular cpn60-specific
polypeptide
may be employed, either alone or in conjunction with a universal antibody;
such
antibodies are referred to as "specific" antibodies herein. The universal and
specific
25 antibodies may be employed simultaneously or in series. For example, a
universal
antibody may be used as a first screen to determine the presence or absence of
a cpn60-
specific polypeptide. Subsequently, a specific antibody, such as one specific
for a cpn60-
specific polypeptide of a particular microbe, e.g., Campylobacter jejurai, may
be
employed. In such assays, monoclonal antibodies may be particularly useful
(e.g.,
3o sensitive) to identify cpn60-specific polypeptides of a particular microbe.
W general, a protein of interest (e.g., a cpn60 protein against which one
wishes to
prepare antibodies) is produced recombinantly, by chemical synthesis, or by
purification
of the native protein, and then used to immunize animals. As used herein, an
intact cpn60
protein rnay be employed, or a cpn60-specific polypeptide may be employed,
provided
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
that the cpn60-specific polypeptide is capable of generating the desired
immune response.
See, for example, WO 200265129 for examples of epitopic sequences that bind to
human
antibodies against Chlamydia t~achomatis; such epitopic sequences may be
useful in
generating antibodies against Chlanaydia spp. for use in the present
invention. See also
U.S. Pat. No. 6,497,880, which sets forth nucleic acid sequences; amino acid
sequences,
expression vectors, purified proteins, antibodies, etc. specific to
Aspergillus fumigatus
and Caradida glabrata. Purified Aspe~gillus furnigatus and Candida glabrata
cpn60
proteins, or proteolytically or synthetically generated fragments thereof, can
be used to
immunize animals to generate antibodies for use in the methods of the present
invention.
o Finally, see WO 200257784, disclosing substantially purified Clalanaydia
hsp60 (cpn60)
polypeptides. Such polypeptides may also be used to generate antibodies for
use in the
methods of the present invention.
As discussed previously, one may wish to prepare universal or specific
antibodies
to cpn60 proteins or polypeptides. A cpn60-specific polypeptide may be used to
generate
~5 a universal antibody, for example, if it maintains an epitope that is
common to at least two
cpn60 proteins, or, e.g., to all cpn60 proteins that one wishes to detect
(e.g., the cpn60
proteins of the Campylobacter genera). Alternatively, a cpn60 protein or cpn60-
specif c
polypeptide rnay be used to generate antibodies specific for a particular
cpn60 protein or
polypeptide present in a particular microbe, e.g., only Campylobacte~ jejuni.
2o Various host animals including, for example, rabbits, chickens, mice,
guinea pigs,
and rats, can be immunized by inj ection of the protein of interest. Adjuvants
can be used
to increase the immunological response depending on the host species and
include
Freund's adjuvant (complete and incomplete), mineral gels such as aluminum
hydroxide,
surface active substances such as lysolecithin, platonic polyols, polyanions,
peptides, oil
25 emulsions, keyhole limpet hemocyanin (KLH), and dinitrophenol. Polyclonal
antibodies
are heterogenous populations of antibody molecules that are specific for a
particular
antigen, which are contained in the sera of the immunized animals. Monoclonal
antibodies, which are homogeneous populations of antibodies to a particular
epitope
contained within an antigen, can be prepared using standard hybridoma
technology. In
3o particular, monoclonal antibodies can be obtained by any technique that
provides for the
production of antibody molecules by continuous cell lines in culture such as
described by
Kohler, G. et al., Nature, 1975, 256:495, the human B-cell hybridoma technique
(Kosbor
et al., Immunology Today, 1983, 4:72; Cole et al., Proc. Natl. Acad. Sci. USA,
1983,
80:2026), and the EBV-hybridoma technique (Cole et al., "Monoclonal Antibodies
and
31
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
Cazzcer Therapy", Alan R. Liss, Inc., 1983, pp. 77-96). Such antibodies can be
of any
immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass
thereof. The
hybridoma producing the monoclonal antibodies of the invention can be
cultivated in
vitro or in vivo.
A chimeric antibody is a molecule in which different portions are derived from
different animal species, such as those having a variable region derived from
a marine
monoclonal antibody and a human immunoglobulin constant region. Chimeric
antibodies
can be produced through standard techniques.
Antibody fragments that have specific binding affinity for a cpn60-specific
polypeptide can be generated by known techniques. For example, such fragments
include, but are not limited to, F(ab')2 fragments that can be produced by
pepsin digestion
of the antibody molecule, and Fab fragments that can be' generated by reducing
the
disulfide bridges of F(ab')2 fragments. Alternatively, Fab expression
libraries can be
constructed. See, for example, Huse et al., 1989, Sciefzce, 246:1275. Single
chain Fv
~ 5 antibody fragments are formed by linking the heavy and light chain
fragments of the Fv
region via an amino acid bridge (e.g., 15 to 18 amino acids), resulting in a
single chain
polypeptide. Single chain Fv antibody fragments can be produced through
standard
techniques. See, for example, U.S. Patent No. 4,946,778. '
Once produced, antibodies or fragments thereof are tested for recognition of a
2o cpn60 protein or cpn60-specific polypeptide by standard immunoassay methods
including, for example, ELISA techniques, countercurrent immuno-
electrophoresis
(CIEP), radioimmunassays (RIA), radioimmunoprecipitations, dot blots,
inhibition or
competition assays, sandwich assays, immunostick (dipstick) assays,
immunochromatographic assays, immunofiltration assays, latex beat
agglutination assays,
25 immunofluoroescent assays, biosensor assays. See, Short Protocols in
Molecular
Biology, Chapter 11, Green Publishing Associates and John Wiley & Sons, Edited
by
Ausubel, F.M et al., 1992; Antibodies: A Laboratory Manual, Harlow and Lane
(eds.),
Cold Spring Harbor Laboratory Press, 1988; and U.S. Pat. Nos. 4,376,110;
4,486,530;
and 6,497,880. Antibodies or fragments can also be tested for their ability to
react
3o universally, e.g., with two or more cpn60 proteins or cpn60-specific
polypeptides, such as
a subset of cpn60 proteins and polypeptides (e.g., the cpn60 proteins from a
bacterial
genera such as Cl~stridium), or specifically with a particular cpn60 protein
(e.g., the
cpn60 protein of Clostridium perfrizzgezzs).
32
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
In antibody assays, the antibody itself or a secondary antibody that binds to
it can
be detectably labeled. Alternatively, the antibody can be conjugated with
biotin, and
detectably labeled avidin (a protein that binds to biotin) can be used to
detect the presence
of the biotinylated antibody. Combinations of these approaches (including
"mufti-layer"
assays) familiar to those in the art can be used to enhance the sensitivity of
assays. Some
of these assays (e.g., immunohistological methods or fluorescence flow
cytometry) can be
applied to histological sections or unlysed cell suspensions. The methods
described
below for detecting a cpn60-specific polypeptide in a liquid sample can also
be used to
detect a cpn60-specific polypeptide in cell lysates.
Methods of detecting a cpn60-specific polypeptide in a liquid sample generally
involve contacting a sample of interest with an antibody that binds to a cpn60-
specific
polypeptide and testing for binding of the antibody to a component of the
sample. In such
assays the antibody need not be detectably labeled and can be used without a
second
antibody that binds to a cpn60-specific polypeptide. For example, an antibody
specific
for a cpn60-specific polypeptide may be bound to an appropriate solid
substrate and then
exposed to the sample. Binding of a cpn60-specific polypeptide to the antibody
on.the
solid substrate may be detected by exploiting the phenomenon of surface
plasmon
resonance, which results in a change in the intensity of surface plasmon
resonance upon
binding that can be detected qualitatively or quantitatively by an appropriate
instrument,
2o e.g., a Biacore apparatus (Biacore International AB, Rapsgatan, Sweden).
Moreover, assays for detection of a cpn60-specific polypeptide in a liquid
sample
can involve the use, for example, of (a) a single antibody specific for a
cpn60-specific
polypeptide that is detectably labeled; (b) an unlabeled antibody that is
specific for a
cpn60-specific polypeptide and a detectably labeled secondary antibody; or (c)
a
biotinylated antibody specific for a cpn60-specific polypeptide and detectably
labeled
avidin. In addition, combinations of these approaches (including "mufti-layer"
assays)
familiar to those in the art can be used to enhance the sensitivity of assays.
In these
assays, the sample or an aliquot of the sample suspected of containing a
microbe can be
immobilized on a solid substrate, such as a nylon or nitrocellulose membrane,
by, for
3o example, "spotting" an aliquot of the liquid sample or by blotting of an
electrophoretic gel
on which the sample or an aliquot of the sample has been subjected to
electrophoretic
separation. The presence or amount of cpn60-specific polypeptide on the solid
substrate
is then assayed using any of the above-described forms of the cpn60-specific
polypeptide
33
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
specific antibody and, where required, appropriate detectably labeled
secondary
antibodies or avidin.
The invention also features "sandwich" assays. In these sandwich assays,
instead
of immobilizing samples on solid substrates by the methods described above,
any cpn60
specific polypeptide that may be present in a sample can be immobilized on the
solid
substrate by, prior to exposing the solid substrate to the sample, conjugating
a second
("capture") antibody (polyclonal or mAb) specific for a cpn60-specific
polypeptide to the
solid substrate by any of a variety of methods known in the art. In exposing
the sample to
the solid substrate with the second antibody specific for cpn60-specific
polypeptide
o bound to it, any cpn60-specific polypeptide in the sample (or sample
aliquot) will bind to
the second antibody on the solid substrate. The presence or amount of cpn60-
specific
polypeptide bound to the conjugated second antibody is then assayed using a
"detection"
antibody specific for a cpn60-specific polypeptide by methods essentially the
same as
those described above using a single antibody specific for a cpn60-specific
polypeptide.
It is understood that in these sandwich assays, the capture antibody should
not bind to the
same epitope (or range of epitopes in the case of a polyclonal antibody) as
the detection
antibody. Thus, if a mAb is used as a capture antibody, the detection antibody
can be
either: (a) another mAb that binds to an epitope that is either completely
physically
separated from or only partially overlaps with the epitope to which the
capture mAb
2o binds; or (b) a polyclonal antibody that binds to epitopes other than or in
addition to that
to which the capture mAb binds. On the other hand, if a polyclonal antibody is
used as a
capture antibody, the detection antibody can be either (a) a mAb that binds to
an epitope
to that is either completely physically separated from or partially overlaps
with any of the
epitopes to which the capture polyclonal antibody binds; or (b) a polyclonal
antibody that
binds to epitopes other than or in addition to that to which the capture
polyclonal antibody
binds. Assays that involve the used of a capture and detection antibody
include sandwich
ELISA assays, sandwich Western blotting assays, and sandwich immunomagnetic
detection assays.
Suitable solid substrates to which the capture antibody can be bound include,
3o without limitation, the plastic bottoms and sides of wells of microtiter
plates, membranes
such as nylon or nitrocellulose membranes, and polymeric (e.g., without
limitation,
agarose, cellulose, or polyacrylamide) beads or particles. It is noted that
antibodies bound
to such beads or particles can also be used for immunoaffinity purification of
cpn60-
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WO 2004/051226 PCT/US2003/038814
specific polypeptides. Dipstick/immunostick formats can employ a solid phase,
e.g.,
polystyrene, paddle or dispstick.
Methods of detecting or for quantifying a detectable label depend on the
nature of
the label and are known in the art. Appropriate labels include, without
limitation,
radionuclides (e.g., lash isih 355 3H~ 32P~ ssp~ or 14C), fluorescent moieties
(e.g.,
fluorescein, rhodamine, or phycoerythrin), luminescent moieties (e.g., QdotTM
nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, CA),
compounds that
absorb light of a defined wavelength, or enzymes (e.g., alkaline phosphatase
or
horseradish peroxidase). The products of reactions catalyzed by appropriate
enzymes can
1 o be, without limitation, fluorescent, luminescent, or radioactive, or they
may absorb visible
or ultraviolet light. Examples of detectors include, without limitation, x-ray
film,
radioactivity counters, scintillation counters, spectrophotometers,
colorimeters,
fluorometers, luminometers, and densitometers.
The methods of the present invention may employ a control sample. In assays to
~5 detect the presence or absence of a microbe, the concentration of a cpn60-
specific
polypeptide in, for example, a food sample suspected of bei.ngcontaminated, or
at risk of
being contaminated, with a microbe may be compared to a control sa~Tiple,
e.g., a food
sample known not to be infected. The control sample may be taken from the same
high-
risk environment, e.g., in a different. location known to be uncontaminated,
or can be a
2o control sample taken from a non-high-risk environment. Alternatively, the
control
sample may be taken from the same location of a high-risk environment but at
an earlier
or later time-point when the location was known to,be uncontaminated. A
significantly
higher concentration of cpn60-specific polypeptide in the suspect sample
relative to the
control sample would indicate the presence of a microbe.
25 It is understood that, while the above descriptions of the diagnostic
assays may
refer to assays on food samples or bodily fluid samples, the assays can also
be carried out
on any of the other fluid or solubilized samples listed herein, such as water
samples or
buffer samples (e.g., buffer used to extract a sample from a fomite).
30 ~the~ polypeptide detection assays
The present invention also contemplates the use of other analytical techniques
for
detecting cpn60-specific polypeptides. Recent analytical instrumentation and
methodology advances that have arisen in the context of proteomics research
are
applicable in the methods of the present invention. See, generally, PR
Jungblut,
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
"Proteome Analysis of Bacterial Pathogens," Micnobes & Isafection 3 (2001):
831-840; G
MacBeath and SL Schreiber, "Printing Proteins as Microarrays for High-
Throughput
Function Determination," Science 289 (2000): 1760-1763; T Madoz-Gdrpide, H
Wang,
and DE Misek, "Protein-Based Microarrays: A Tool for Probing the Proteome of
Cancer
Cells and Tissues," PYOteonaics 1 (2001): 1279-1287; S Patterson, "Mass
Spectrometry
and Proteomics," Playsiological Genomics 2 (2000): 59-65; and A Schevchenko et
al.,
"Maldi Quadrupole Time-of Flight Mass Spectrometry: A Powerful Tool for
Proteomic
Research," Analytical Claemistry 72 (2000):2132-2141.
Mass-spectrophotometric techniques have been increasingly used to detect and
1 o identify proteins and protein fragments at low levels, e.g., finol or
pmol. Mass
spectrometry has become a major analytical tool for protein and proteomics
research
because of advancements in the instrumentation used for biomolecular
ionization,
electrospray ionization (ESI), and matrix-assisted laser desorption-ionization
(MALDI).
MALDI is usually combined with a time-of flight (TOF) mass analyzer.
Typically, 0.5
~ 5 ~ 1 of sample that contains 1-10 pmol of protein or peptide is mixed with
an equal
volurrie of a saturated matrix solution and allowed to dry, resulting in the
co-
crystallization of the analyte with the matrix. Matrix compounds that are used
include
sinapic acid and a-hydroxycinnarnic acid. The cocrystallized material on the
target plate
is irradiated with a nitrogen laser pulse, e.g., at a wavelength of 337 nm, to
volatilize and
2o ionize the protein or peptide molecules. A strong acceleration field is
switched on, and
the ionized molecules move down the flight tube to a detector. The amount of
time
required to reach the detector is related to the mass-to-charge ratio.
Proteolytic mass
mapping and tandem mass spectrometry, when combined with searches of protein
and
protein fragment databases, can also be employed to detect and identify cpn60-
specific
25 polypeptides. See, for example, Devin M. Downard, "Contributions of mass
spectrometry to structural immunology," J. Mass. Spectnom. 35:493-503(2000).
Biomolecular interaction analysis mass spectrometry (BIA-MS) is another
suitable
technique for detecting interactions between cpn60-specific polynucleotides
and cpn60
antibodies. This technology detects molecules bound to a ligand that is
covalently
3o attached to a surface. As the density of biomaterial on the surface
increases, changes
occur in the refractive index at the solution or surface interface. This
change in the
refractive index is detected by varying the angle or wavelength at which the
incident light
is absorbed at the surface. The difference in the angle or wavelength is
proportional to
the amount of material bound on the surface, giving rise to a signal that is
termed surface
36
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
plasmon resonance (SPR), as discussed previously. See, for example, RW Nelson
et al.,
"BIA/MS of Epitope-Tagged Peptides Directly from E. coli Lysate: Multiplex
Detection
and Protein Identification at Low-Femtomole to Subfemtomole Levels,"
Analytical
Chemistry 71 (1999): 2858-2865; see also D Nedelkov and RW Nelson, "Analysis
of
Native Proteins from Biological Fluids by Biomolecular Interaction Analysis
Mass
Spectrometry (BIA/MS): Exploring the Limit of Detection, Identification of Non-
Specific
Binding and Detection of Multiprotein Complexes," Biosensors and
Bioelectronics 16
(2001): 1071-1078.
The SPR biosensing technology has been combined with MALDI-TOF mass
o spectrometry for the desorption and identification of biomolecules. In a
chip-based
approach to BIA-MS, a ligand , e.g., a cpn60 antibody, is covalently
immobilized on the
surface of a chip. A tryptic digest of solubilized proteins from a sample is
routed over the
chip, and the relevant peptides, e.g., cpn60-specific polypeptides, are bound
by the ligand.
After a washing step, the eluted peptides are analyzed by MALDI-TOF mass
spectrometry. The system may be a fully automated process and is applicable to
detecting and characterizing proteins present in complex biological fluids and
cell
extracts at low- to subfemtomol levels.
Mass spectrometers useful for such applications are available from Applied
Biosystems (Foster City, CA); Bruker Daltronics (Billerica, MA) and Amersham
2o Pharmacia (Sunnyvale, CA).
Other suitable techniques for use in the present invention include
"Multidimensional Protein Identification Technologies.". Cells are
fractionally
solubilized and digested, e.g., sequentially with endoproteinase Lys-C and
immobilized
trypsin. The samples are then subj ected to multidimensional protein
identification
technology (MUDPIT), which involves a sequential separation of the peptide
fragments
by on-line biphasic microcapillary chromatography (e.g., strong ion exchange,
then C-18
separation), followed by tandem mass spectrometry (MS-MS). See, for example,
MP
Washburn, D Wolter, and JR Yates, "Large-Scale Analysis of the Yeast Proteome
by
Multidimensional Protein Identification Technology," Nature Biotechnology 19
(2001):
so 242-247.
Articles of Manufacture
The invention also provides articles of manufacture. Articles of manufacture
can
include at least one cpn60 oligonucleotide primer, as well as instructions for
using the
37
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
cpzz60 oligonucleotide(s) to identify and quantify the amount of one or more
microbial
organisms in a biological or non-biological sample.
In one embodiment, the cpn60 oligonucleotide(s) are attached to a microarray
(e.g., a GeneChip°°, Affymetrix, Santa Clara, CA). In another
embodiment, an article of
manufacture can include one or more cpfz60 oligonucleotide primers and one or
more
cpzz60 oligonucleotide probes. Such cpzz.60 primers and probes can be used,
for example,
in real-time amplification reactions to amplify and simultaneously detect
cpn60
amplification products.
Suitable oligonucleotide primers include those that are complementary to
highly
1o conserved regions of cpzz60 and that flank a variable region. Such
universal cpzz60
primers can be used to specifically amplify these variable regions, thereby
providing a
sequence with which to identify microorganisms. Examples of cpn60
oligonucleotide
primers include the following:
5'-GAIIIIGCIGGIGA(T/C)GGIACIACIAC-3' (SEQ ~ N0:6); and
5'-(T/C)(T/G)I(T/C)(T/G)ITCICC(A/G)AAICCIGGIGC(T/C)TT-3' (SEQ ID
N0:7).
Suitable oligonucleotide primers also include those that are complementary to
species-specific cpn60 sequences, and thus result in an amplification product
only if a
particular species is present in the sample.
2o Similar to cpn60 oligonucleotide primers, cph60 oligonucleotide probes
generally
are complementary to cpn60 sequences. cprz60 oligonucleotide probes can be
designed to
hybridize universally to cpn60 sequences, or can be designed for species-
specific
hybridization to the variable region of cpn60 sequences.
An article of manufacture of the invention can further include additional
components for carrying out amplification reactions and/or reactions, for
example, on a
microarray. Articles of manufacture for use with PCR reactions can include
nucleotide
triphosphates, an appropriate buffer, and a polymerase. An article of
manufacture of the
invention also can include appropriate reagents for detecting amplification
products. For
example, an article of manufacture can include one or more restriction enzymes
for
3o distinguishing amplification products from different species of
microorganism, or can
include fluorophore-labeled oligonucleotide probes for real-time detection of
amplification products.
It will be appreciated by those of ordinary skill in the art that different
articles of
manufacture can be provided to evaluate microbes in different types of high-
risk
38
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
environments. For example, a hospital will have a different community of
microbes than
that of a restaurant. Therefore, an article of manufacture designed to
evaluate the
microbes in a hospital may have a different set of controls or a different set
of species-
specific hybridization probes than that designed for a restaurant.
Alternatively, a more
generalized article of manufacture can be used to evaluate the microbes in a
number of
different high-risk environments.
Articles of manufacture also can include at least one cpn60 antibody, as well
as
instructions for using the same to detect the presence of a microbe, and
optionally to
evaluate a microbial profile, in a biological or non-biological sample.
o In one embodiment, one or more cpn60 antibodies are attached to a microarray
(e.g., a 96-microwell plate). For example, a microarray format may include a
variety of
universal and specific cpn60 capture antibodies; the universal and specific
antibodies may
each be located at a different well location. The article of manufacture may
also include
the appropriate detection antibodies, if necessary, and appropriate reagents
for detection
~ 5 of binding of a cpn60-specific polypeptide to one or more capture
antibodies (e.g.,
enzymes, substrates, buffers, and controls).
In another embodiment, an article of manufacture can. include one or more
cpn60
antibodies attached to a dipstick. Such dipsticks can be used, for example, to
detect
cpn60-specific polypeptides in a liquid sample.
EXAMPLES
Example 1- Ouantitatin~Lmicrobial organisms using universal primers and a
universal
rp obe
A biological sample is obtained from poultry GIT and genomic DNA is extracted
using standard methods (Diagnostic Molecular Microbiology: Principles artd
Applications (supra)). Real-time PCR is conducted using universal cpn60
primers having
the nucleotide sequences set forth in SEQ ID N0:6 and SEQ ID N0:7, and a
universal
cpn60 probe having the sequence 5'-GACAAAGTCGGTAAAGAAGGCGTTATCA-3'
(SEQ ll~ N0:8), labeled at the 5' end with fluorescein (fluorophore; Molecular
Probes,
3o Inc.) and at the 3' end with dabcyl (quencher; (4-(4'-
dimethylaminophenylazo)benzoic
acid) succinimidyl ester; Molecular Probes, Inc.). This probe binds to a
variable region of
the cpn60 gene from numerous microbial species; thus the Tm of the probe from
an
amplification product varies depending upon the nucleotide sequence within the
amplification product to which the probe hybridizes.
39
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
The PCR reaction contains 3 TL extracted DNA, 1 TM each universal cpn60
primer, 340 nM universal cpn60 probe, 2.5 units Amplitaq Gold DNA polymerase
(Perkin Elmer), 0.25 mM each deoxyribonucleotide, 3.5 mM MgClz, 50 mM KCI, and
10
mM Tris-HCl, pH 8.0 in a total reaction volume of 50 TL. PCR conditions
include an
initial incubation at 95°C for 10 minutes to activate the Amplitaq Gold
DNA polymerise,
followed by 40 cycles of 30 seconds at 95°C, 60 seconds at 50°C,
and 30 seconds at
72°C. Fluorescence is monitored during the 50°C annealing steps
throughout the 40
cycles. After the cycling steps are complete, the melting temperature of the
universal
probe from the amplification products is analyzed. As the temperature is
increased, the
universal probe is released from the amplification product from each species'
cpn60
sequence at a specific temperature, corresponding to the Tm of the universal
probe and
the cpn60 sequence of the particular species. The step-wise loss of
fluorescence at
particular temperatures is used to identify the particular species present,
and the loss in
fluorescence of each step compared to the total amount of fluorescence
correlates with the
15 relative amount of each microorganism.
E_ xample 2 - Ouantification of microbial or ,anisrtis using universal ors and
species-
specific probes
A biological sample is obtained from poultry GIT and genomic DNA is extracted
2o using standard methods (Diagnostic Molecular- MicYObiology: Principles and
Applications (supra)). Real-time PCR is conducted using universal cpn60
primers having
the nucleotide sequences set forth in SEQ ID N0:6 and SEQ ID NO:7, and species-
specific probes having the nucleotide sequences:
5'-AGCCGTTGCAAAAGCAGGCAAACCGC-3' (SEQ ID NO:9);
25 5'-TTGAGCAAATAGTTCAAGCAGGTAA-3' (SEQ ID NO:10);
5'- GCAACTCTGGTTGTTAACACCATGC-3' (SEQ ID NO:l 1);
5'-TGGAGAAGGTCATCCAGGCCAACGC-3' (SEQ ID N0:12); and
5'- TAGAACAAATTCA,A.AAAACAGGCAA-3' (SEQ ID N0:13).
These species-specific probes hybridize to cpra60 nucleotide sequences from S.
3o enterica, C. perfriragens, E. coli, S. coelicolor-, and C. jejuni,
respectively. The sequences
of the probes are identified by aligning Cpra6O cDNA sequences from the five
organisms
and identifying a sequence that is specific to each particular organism (i.e.,
a sequence not
found in the other organisms). Each of the species-specific probes is labeled
with a
different fluorescent moiety to allow differential detection of the various
species.
CA 02507398 2005-05-25
WO 2004/051226 PCT/US2003/038814
The PCR reaction contains 3 TL extracted DNA, 1 TM each universal cpn60
primer, 340 nM universal cph60 probe, 2.5 units Amplitaq Gold DNA polymerase
(Perkin Elmer), 0.25 mM each deoxyribonucleotide, 3.5 mM MgCl2, 50 mM KCI, and
10
mM Tris-HCI, pH 8.0 in a total reaction volume of 50 TL. PCR conditions
include an
initial incubation at 95°C for 10 minutes to activate the Amplitaq Gold
DNA polymerase,
followed by 40 cycles of 30 seconds at 95°C, 60 seconds at 50°C,
and 30 seconds at
72°C. Fluorescence is monitored during the 50°C annealing steps
throughout the 40
cycles, at wavelengths corresponding to the particular moieties on the probes.
The
amount of fluorescence detected at each of the monitored wavelengths
correlates with the
1o amount of each cpn60 amplification product. The amount of each species-
specific
amplification product is then correlated with the amount of each species of
microbe by
comparison to the amount of amplification product generated from samples
containing
nucleic acid isolated from known amounts of each microbial species.
~ 5 Example 3 - D~sticlc ELISA assay,for Streptococcus
A polystyrene dipstick containing two horizontal bands is constructed: tine
band
consists of broadly r°eactive, polyclonal capture antibodies against
cpn60 proteins from
Sts°eptococcus spp., while the other band is an internal control
consisting of horseradish
peroxidase. The assay is performed by making serial dilutions (1:2, 1:5, 1:10,
etc.) of a
20 liquid sample taken from a high risk environment (e.g., a urine sample or a
blood sample)
directly into a detection reagent and incubating a wetted dipstick in these
dilutions for~5
minutes, and then adding an indicator to detect binding of cpn60 proteins to
the capture
(and detection) antibodies. The detection reagent includes a suitable buffer
and
secondary cpn60 Streptococcus detection antibodies labeled with horseradish
peroxidase.
25 The indicator can be a chromogenic horseradish peroxidase substrate, such
as 2,2'-
AZINO-bis 3-ethylbenziazoline-6-sulfonic acid, or ABTS. ABTS is considered a
safe,
sensitive substrate for horseradish peroxidase that produces a blue-green
color upon
enzymatic activity that can be quantitated at 405-410 run. At the end of the
incubation
and indicator steps, the dipstick is rinsed with water (e.g., deionized water)
and examined
3o for staining of the antibody band by visual inspection. Staining of the
antibody band
reveals the presence of Streptococcus spp. in the sample. The internal control
band
provides a check on the integrity of the detection reagent.
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OTHER EMBODIMENTS
It is to be understood that while the invention has been described in
conjunction
with the detailed description thereof, the foregoing description is intended
to illustrate and
not limit the scope of the invention, which is defined by the scope of the
appended claims.
Other aspects, advantages, and modifications are within the scope of the
following
claims.
42
