WO2004111186A2 - Surveillance et detection de microbes utilises en bioterrorisme - Google Patents

Surveillance et detection de microbes utilises en bioterrorisme Download PDF

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Publication number
WO2004111186A2
WO2004111186A2 PCT/US2003/038020 US0338020W WO2004111186A2 WO 2004111186 A2 WO2004111186 A2 WO 2004111186A2 US 0338020 W US0338020 W US 0338020W WO 2004111186 A2 WO2004111186 A2 WO 2004111186A2
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WIPO (PCT)
Prior art keywords
sample
bioterrorism
cpn60
cpnόo
microbe
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PCT/US2003/038020
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English (en)
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WO2004111186A3 (fr
Inventor
Wade Robey
Alison Jones
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Cargill, Incorporated
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Priority claimed from US10/306,113 external-priority patent/US20040101860A1/en
Priority claimed from US10/449,460 external-priority patent/US20040259226A1/en
Application filed by Cargill, Incorporated filed Critical Cargill, Incorporated
Priority to AU2003304213A priority Critical patent/AU2003304213A1/en
Publication of WO2004111186A2 publication Critical patent/WO2004111186A2/fr
Publication of WO2004111186A3 publication Critical patent/WO2004111186A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • This invention relates to monitoring for and detecting microbes, specifically those microbes used in bioterrorism, by detecting microbial markers.
  • Bioterrorism can be defined as the use, or threatened use, of biological agents to promote or spread fear or intimidation upon an individual, a specific group, or the population as a whole for religious, political, ideological, financial, or personal purposes.
  • the biological agents generally used in bioterrorism are typically found in nature, but can be weaponized to enhance their virulence in humans and make them resistant to vaccines and antibiotics. Weaponizing can involve using selective pressure or genetic engineering to mutate or modify the genetic composition of the agent.
  • Bioterrorism agents may be disseminated by various methods, including aerosolization, through specific blood-feeding insects, or by contaminating food or water supplies. Representative microbes that are used as biological agents in bioterrorism include the following. Anthrax
  • Bacillus anthracis the organism that causes anthrax, derives its name from the Greek word for coal, anthracis, because of its ability to cause black, coal-like cutaneous eschars.
  • anthrax was developed as part of a larger biological weapons program by several countries, including the Soviet Union and the U.S. The number of nations believed to have biological weapons programs has steadily risen from 10 in 1989 to 17 in 1995, but how many nations are working with anthrax is uncertain. Perhaps more insidious is the specter of autonomous groups with ill intentions of using anthrax in acts of terrorism.
  • Botulinum toxin is derived from Clostridium botulinum. Seven antigenic types of C. botulinum toxin exist, designated A through G. They can be identified based on antibody cross reactivity studies, e.g., anti-A toxin antibodies do not neutralize the B through G toxins. Botulinum toxin poses a major threat because of its extreme potency and lethality; its ease of production, transport and misuse; and the potential need for prolonged intensive care in affected persons. Botulinum toxin is the single most poisonous substance known. A number of countries and groups have developed or are developing botulinum toxin as a biological weapon. Aum Shinrikyo tried but failed to use botulinum toxin as a biological weapon.
  • Naturally occurring botulism results from the absorption of botulinum toxin into the circulation through the mucosal surface ⁇ e.g., the gut, or a lung) or a wound.
  • Botulinum toxin does not penetrate intact skin.
  • the toxin irreversibly binds to peripheral cholinergic synapses, preventing the release of the neurotransmitter acetylcholine from the terminal end of motor neurons. This leads to muscle paralysis, and in severe cases, can lead to a need for mechanical respiration.
  • the incubation period for food-borne botulism can be from 2 hours to 8 days after ingestion, depending on the dose of the bacteria or the toxin.
  • the average incubation period is 12-72 hours after ingestion.
  • Patients with botulism typically present with difficulty speaking, seeing and/or swallowing.
  • Prominent neurologic findings in all forms of botulism include ptsosis, diplopia, blurred vision, dysarthria and dysphagia.
  • Patients typically are afebrile and do not have an altered level of consciousness. Patients may initially present with gastrointestinal distress, nausea, and vomiting that precede the neurological symptoms. Symptoms are similar for all toxin types, but the severity of illness can vary widely, in part depending on the amount of toxin absorbed.
  • the second plague pandemic also known as the Black Death or the Great Pestilence, erupted and within 5 years, had ravaged the Middle East and killed more than 13 million in China and 20-30 million (one third of the population) in Europe.
  • Plague is one of very few diseases that can create widespread panic following the discovery of even a small number of cases. This was apparent in Surat, India in 1994, when an estimated 500,000 persons fled the city in fear of a plague epidemic.
  • the U.S. and Soviet biological weapons programs developed techniques to directly aerosolize plague particles, a technique that leads to pneumonic plague, an otherwise uncommon, highly lethal and potentially contagious form of plague. A modern bioterrorism attack would most probably occur via aerosol dissemination of Y.
  • Francisella tularensis the organism that causes tularemia, is one of the most infectious pathogenic bacteria known, requiring inoculation or inhalation of as few as 10 organisms to cause disease. It is considered to be a dangerous potential biological weapon because of its extreme infectivity, ease of dissemination, and substantial capacity to cause illness and death.
  • F. tularensis as a biological weapon was studied by the Japanese as well as by the U.S. and its allies.
  • Tularemia was one of several biological weapons that were stockpiled by the U.S. military in the late 1960's, all of which were destroyed by 1973.
  • the Soviet Union continued weapons production of antibiotic and vaccine-resistant strains into the early 1990s.
  • F. tularensis is a hardy, non-spore forming organism that is capable of surviving for weeks at low temperatures in water, moist soil, hay, straw or decaying animal carcasses.
  • F. tularensis has been divided into two subspecies: F. tularensis biovar tularensis (type A), which is the most common biovar isolated in North America and may be highly virulent in humans and animals; and F. tularensis biovar palaearctica (type B) which is relatively avirulent and thought to the cause of all human tularemia in Europe and Asia.
  • a WHO expert committee reported in 1970 that if 50 kg of virulent F.
  • tularensis was dispersed as an aerosol over a metropolitan area with a population of 5 million, there would an estimated 250,000 incapacitating casualties, including 19,000 deaths. Aerosol dissemination of F. tularensis in a populated area would be expected to result in the abrupt onset of large numbers of cases of acute, non-specific febrile illness beginning 3 to 5 days later (incubation range, 1-14 days), with pleuropneumonitis developing in a significant proportion of cases over the ensuing days and weeks. Without antibiotic treatment, the clinical course could progress to respiratory failure, shock and death.
  • microbes that may not have a lethal effect can be used as biological agents in bioterrorism.
  • Such microbes include E. coli, or Salmonella spp., which could be used to infect a large population of individuals and make them sick.
  • Salmonella spp. which could be used to infect a large population of individuals and make them sick.
  • followers of Bhagwan Shree Rajneesh contaminated numerous salad bars in restaurants with Salmonella, resulting in 751 people becoming ill.
  • microbes that can be used in agricultural bioterrorism such as Liberobacter africanus, Liberobacter asiaticus, Peronosclerospora philippinesis, Phakospsora pachyrhizi, Ralstonia solanacearum, race 3, biovar 2, Sclerophthora rayssiae var. zeae, Synchytrium endobioticum, Xanthomonas oryzae pv. oryzicola, and Xylella fastidiosa (citrus variegated chlorosis strain). See, for example, the Agriculture Bioterrorism Protection Act of 2002 (7 C.F.R. ⁇ 331). Therefore, a number of microbes can be used in bioterrorism.
  • the invention is based on the discovery that the presence or absence of a microbe used in bioterrorism can be determined quickly and sensitively by detecting the presence and/or concentration of a microbial marker, specifically a cpn ⁇ O marker, in a sample.
  • Chaperonin 60 (cpn ⁇ O) markers are particularly useful for determining the presence of a microbe in a sample and for optionally identifying the microbe.
  • Chaperonin proteins are molecular chaperones required for proper folding of polypeptides in vivo.
  • cpn ⁇ O is found universally in prokaryotes and in the organelles of eukaryotes, and can be used as a species-specific target and/or probe for identification and classification of microbes.
  • the invention provides a method for detecting the presence, absence, or amount of one or more microbes used in bioterrorism.
  • a method for detecting the presence, absence, or amount of one or more microbes used in bioterrorism includes a) providing a sample obtained from an environment susceptible to bioterrorism attack or an environment within which a bioterrorism attack has taken place; and b) detecting the presence, absence, or amount oiz cpn ⁇ O marker in the sample, wherein the cpn ⁇ O marker is specific for one or more of the microbes used in bioterrorism.
  • the presence of the cpn ⁇ O marker in the sample is indicative of the presence, in the environment, of one or more of the microbes used in bioterrorism.
  • the detecting step can be capable of identifying the microbe(s) in the sample, and further can be capable of quantitating the microbe(s) in the sample.
  • the detecting step can be a nucleic acid-based assay such as PCR or FISH assays.
  • the detecting step also or alternatively can be a polypeptide-based assay such as an irnmunodiagnostic assay (e.g., ELISA), a mass spectrometric technique, or a surface plasmon resonance technique.
  • the sample is provided at two or more points, which can be time points or location points.
  • the method of the invention can further include comparing the presence, absence, or amount of the microbe at the two or more points.
  • the method of the invention can further include acquiring a control sample from one or both of the environments.
  • the method of the invention can still further include comparing the presence, absence, or amount of microbes used in bioterrorism in the control sample with the presence, absence, or amount of the microbes used in bioterrorism in the sample.
  • the cpn ⁇ O marker is a cpn ⁇ O nucleic acid or a cpn60 polypeptide.
  • the cpn ⁇ O marker is a cpn ⁇ O nucleic acid
  • the cpn ⁇ O nucleic acid can be a nucleic acid coding sequence of a cpn ⁇ O protein.
  • the cpn ⁇ O nucleic acid is an amplified sequence of a cpn ⁇ O coding sequence from the microbes used in bioterrorism.
  • microbes used in bioterrorism and that can be detected by the method of the invention include B. anthracis, C. botulinum, Y. pestis, and F. tularensis.
  • Representative environments susceptible to bioterrorism attack can include a food-service facility, a water facility, a transportation facility, an entertainment facility, a shopping mall, and an office building.
  • a food-service facility include a restaurant, a cafeteria, a snack bar, and a convenience store.
  • Examples of a water facility include potable water facilities, desalinization facilities, dams, recycled water facilities, water storage tanks, and potable water reservoirs.
  • Examples of a transportation facility include an airport, a bus terminal, a train terminal, a port, a Custom's checkpoint, and an immigration checkpoint.
  • Examples of an entertainment facility include a club, a theater, a stadium, and an arena.
  • a sample can be a biological sample or a non-biological sample.
  • Biological samples include water, air, food, a tissue sample from an animal or human, or a fluid sample from an animal or human.
  • Non-biological samples can be collected, for example, from a fomite.
  • the invention provides an article of manufacture that includes at least one cpn ⁇ O antibody and an indicator molecule.
  • the cpn60 antibody has specific binding affinity for a cpn ⁇ O polypeptide from a microbe used in bioterrorism.
  • the cpn ⁇ O antibody is attached to a solid support such as a dipstick.
  • An article of manufacture of the invention can further include instructions for using the cpn ⁇ O antibody to detect a cpn ⁇ O-containing microbe used in bioterrorism.
  • the invention provides an article of manufacture that includes at least one cpn ⁇ O oligonucleotide, and instructions therein for using the cpn ⁇ O oligonucleotide(s) to determine, in a sample, the presence, absence, or amount of a microbe used in bioterrorism.
  • the cpn ⁇ O oligonucleotide is complementary to cpn ⁇ O nucleic acids from at least one microbe used in bioterrorism.
  • One or more cpn60 oligonucleotide(s) can be attached to a microarray in an article of manufacture of the invention. Unless otherwise defined, all technical and scientific terms used herein have
  • FIG. 1 is the sequence of a cpn ⁇ O gene from E. coli (SEQ E) NO:4; GenBank
  • FIG. 2 is the sequence of a cpn60 gene from Salmonella enterica (SEQ ID N0:5; GenBank Accession No. NC_003198). 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 Salmonella enterica (SEQ ID N0:5; 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.
  • the present invention provides methods of monitoring for and detecting the presence or absence of one or more microbes used in bioterrorism.
  • the presence or absence of a microbe used in bioterrorism can be determined quickly and sensitively by detecting the presence and/or concentration of a microbial marker, specifically a cpn ⁇ O marker, in a sample.
  • environments that are susceptible to bioterrorism attack are numerous.
  • environments that are susceptible to bioterrorism attack include but are not limited to the following: a food-service facility, a water facility, a transportation facility, an entertainment facility, a shopping mall, and an office building.
  • the air within such facilities or buildings provides an environment susceptible to bioterrorism attack, as does the atmospheric air outside.
  • Food-service facilities include such places as restaurants, cafeterias, snack bars, and convenience stores.
  • Water facilities can include potable water facilities, desalinization facilities, dams, recycled water facilities, water storage tanks, and potable water reservoirs (e.g., water coolers).
  • Non-limiting examples of transportation facilities include airports, bus terminals, train terminals, ports,
  • a transportation facility also can include the means for transportation used or housed within such a facility, such as a car, a bus, a plane, a train, or a ship.
  • Entertainment facilities can include, without limitation, nightclubs, theaters, stadiums, and arenas. With respect to shopping malls and office buildings, it is likely that the larger facilities (e.g., megamalls, or high rise office buildings) with more incoming and outgoing traffic would present a more desirable target for a bioterrorism attack, although the invention is not limited to facilities having a minimum size or occupancy number.
  • the methods of the invention for detecting the presence, absence, or amount of microbes used in bioterrorism also can be used to evaluate and confirm false or hoax claims of bioterrorism.
  • Methods of the invention for rapidly evaluating claims of bioterrorism that may be false can significantly reduce the panic that may ensue following a claim of bioterrorism, as well as reduce or eliminate the unnecessary mobilization of response and medical teams.
  • the methods described herein are capable of detecting the presence, absence, or amount of a microbe, and optionally identifying the microbe, based on the presence of a cpn60 marker in a sample.
  • a sample can be collected from high-risk areas such as air, food, and transportation and entertainment facilities to monitor for a bioterrorism attack. After a bioterrorism attack has occurred, however, samples can be collected from anything in the vicinity of the attack such as surfaces, objects, and individuals. Although by no means limiting, samples generally can be classified as "biological” and "non-biological.”
  • a biological sample can refer to any sample obtained, directly or indirectly, from an animal or a human.
  • a biological sample also can refer to environmental samples such as those obtained from food (prepared or raw), water, or air.
  • Representative biological samples that can be obtained from an animal or a human include or are derived from biological tissues ⁇ e.g., skin and hair), biological fluids ⁇ e.g., blood, urine, and saliva), and biological elimination products ⁇ e.g., feces).
  • biological tissues can include biopsy samples from, for example, the gastrointestinal tract, the mucous membrane, the mouth, the nose, and the skin, or swabs of the biological tissue of interest, e.g., nasal swabs, throat swabs, dermal swabs.
  • the tissue can be any appropriate tissue from a human, but also can be derived from an animal, such as a cow, a pig, a horse, a goat, a sheep, a dog, a cat, or a bird.
  • 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 lung lavage, or a gastric lavage; an 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 a human or an animal 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).
  • An air or water sample can be collected using known methods and equipment. Air and/or water samples can be collected periodically over time ⁇ e.g., hours, days, weeks, or months) or over multiple regions ⁇ e.g., multiple locations within a city, or multiple locations around a country) to monitor for microbes used in bioterrorism.
  • a biological sample can also be a food sample.
  • 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, or fruits), hi other embodiments, the food sample may be unprocessed and/or raw.
  • the food sample may be perishable.
  • food samples will be taken from food products such as beef, pork, 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.
  • a "non-biological" sample includes any sample taken from, for example, an inanimate object or a solid surface.
  • a fomite may be sampled to detect the presence or absence of a microbe.
  • a fomite is a physical inanimate object onto which a microbe can settle or be deposited.
  • Representative fomites from, for example, an entertainment or a transportation facility include the surface of floors, seats, or serving areas. Additional non-limiting examples of fomites include utensils, drinking glasses, food processing equipment, cutting surfaces, floors, ceilings, walls, drains, ventilation systems, machines, door handles, and clothes.
  • a microbe may be left as a residue on a fomite. In such cases, it is important to accurately detect the presence of the microbe on the fomite.
  • microbes may exist in viable but non-culturable forms on fomites, or that non-culturable bacteria of selected species can be resuscitated to a culturable state under certain conditions. Often such non-culturable bacteria are present in bio films on fomites. Accordingly, detection methods that rely on culturable forms may significantly under-report microbial contamination on fomites.
  • the methods of the present invention specifically PCR-based methods, can aid in the detection of microbes, particularly non-culturable forms, by amplification and detection of cpn ⁇ O nucleic acid sequences.
  • AOAC International Association of Analytical Communities International
  • WO 98/32020 and U.S. Patent No. 5,624,810, which set forth methods and devices for collecting and concentrating microbes from the air, liquid, or a surface.
  • WO 98/32020 also provides methods for removing somatic cells or animal body cells present at varying levels in certain samples.
  • 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.
  • a sample suspected of containing a biological microbe may 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.
  • appropriate filters and/or immunomagnetic separations can concentrate a microbe without the need for an extended growth stage.
  • antibodies specific for a cpn60 polypeptide can be attached to magnetic beads and/or particles. Multiplexed separations in which two or more concentration processes (e.g., centrifugation, membrane filtration, electrophoresis, ion exchange, affinity chromatography, and immunomagnetic separations) are employed also are contemplated.
  • Certain air or water samples may need to be concentrated.
  • certain air sampling methods require the passage of a prescribed volume of air over a filter to trap any microbes, followed by transfer into a buffer or liquid culture.
  • the focused air is passed over a plate (e.g., agar) medium for culturing.
  • a plate e.g., agar
  • Methods for sampling a tissue or a fomite with a swab are known to those of skill in the art.
  • 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.
  • an appropriate buffer such as Cary-Blair medium, Stuart's medium, Amie's medium, PBS, buffered glycerol saline, or water
  • the so-recovered sample may then be analyzed according to the methods described herein for the presence of a microbe used in bioterrorism.
  • the swab may be used to inoculate a liquid or plate (e.g., agar) medium in order to culture of any microbes present.
  • Suitable swabs include both cotton and sponge swabs; see, for example, those provided by Tecra®, such as the Tecra ENVmOSWAB®.
  • samples can be used "as is,” or may need to be treated prior to application of the detection methods employed herein.
  • samples can be processed (e.g., by nucleic acid or protein extraction methods and/or kits known in the art) to release nucleic acid or proteins.
  • a biological sample can be contacted directly with the appropriate reagents (e.g., PCR reaction components and appropriate oligonucleotide primers and/or probes).
  • Detection of cpn ⁇ O Markers Methods provided herein are useful for determining the presence of one or more microbes used in bioterrorism and optionally for identifying the particular microbe.
  • a microbe used in bioterrorism includes B. anthracis, C. botulinum, Y. pestis, and F. tularensis.
  • a microbe in a sample e.g., a biological sample or a non- biological sample obtained from an environment susceptible to a bioterrorism attack or an environment in which a bioterrorism attack has occurred can be determined using methods that involve detection of a cpn60 marker.
  • cpn ⁇ O markers include cpn ⁇ O nucleic acids and cpn ⁇ O polypeptides.
  • a cpn ⁇ O nucleic acid is a nucleic acid that includes, is complementary to, or specifically hybridizes to all or a portion of a cpn.60 nucleic acid sequence.
  • the term "nucleic acid” as used herein encompasses both RNA and DNA, including genomic DNA. Nucleic acid can be double-stranded or single-stranded, and can contain one or more restriction enzyme sites.
  • a cpn ⁇ O nucleic acid marker will be all or a portion of the nucleic acid coding sequence of a cpn ⁇ O protein.
  • a cpn ⁇ O nucleic acid may be specific to a particular species of microbe or may be universal to multiple species of microbe.
  • Species-specific cpn.60 nucleic acid sequences are cpn ⁇ O nucleic acid sequences that hybridize preferentially to cpn ⁇ O nucleic acid sequences from a given microbial species under appropriate assay conditions.
  • oligonucleotides to detect such species-specific cpn ⁇ O nucleic acid sequences by e.g., aligning cpn ⁇ O nucleic acid coding sequences from multiple species and looking for regions within cpn ⁇ O sequences that vary between the species, e.g., sequences from one species that would not cross-hybridize to cpn ⁇ O nucleic acid sequences from other species under the same assay conditions.
  • 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 similarity
  • Use of such species-specific oligonucleotides in the methods described herein allows for the discriminatory detection of particular microbes in a sample.
  • methods of the invention that use species-specific cpn ⁇ O nucleic acids can be used for epidemiology studies of, for example, the origin or source of a microbe used in a bioterrorism attack.
  • cpn ⁇ O primers or probes can be designed that discriminate between particular strains or sub-types within a species of microbe used in bioterrorism based on the presence of mutations or polymorphisms within the cpn ⁇ O sequence.
  • methods of the invention can be used to compare microbes used in a bioterrorism attack with known sources of such microbes (depository's, stocks from countries or political/terrorist groups known to maintain stocks of such microbes used in bioterrorism) to track down the source of a bioterrorism attack.
  • percent sequence identity two sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two 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 arrive 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.
  • 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 up to 79%.
  • 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. gov.
  • BLAST searches can be performed to determine percent sequence identity between a cpn ⁇ O nucleic acid sequence from one species of microbe and a cpn ⁇ O nucleic acid sequence from another species of microbe aligned using the Altschul et al. algorithm.
  • BLASTN is the program used to align and compare the identity between nucleic acid sequences
  • BLASTP is the program used to align and compare the identity between amino acid sequences.
  • Sequences of cpn ⁇ O nucleic acids from microbes used in bioterrorism can be obtained using conventional methods.
  • cpn ⁇ O sequences can be found in public databases such as GenBank.
  • Representative nucleic acid sequences from the genome of microbes using in bioterrorism are shown in GenBank Accession Nos. NC_003366, AP003193, NC_003143, AJ414142, and NC_003995.
  • a representative cpn ⁇ O nucleic acid sequence from B. anthracis is shown in GenBank Accession No. AB028452.
  • cpn ⁇ O nucleic acid sequences from microbes used in bioterrorism can be identified using homologous cpn ⁇ O nucleic acid sequences from other microbes in, for example, BLAST searches of public databases, or hybridization to libraries containing nucleic acids from microbes used in bioterrorism.
  • a "universal" cpn ⁇ O nucleic acid is a cpn ⁇ O nucleic acid sequence that is capable of hybridizing or annealing under the appropriate assay conditions to cpn ⁇ O nucleic acid sequences from more than one species of microbe. Such sequences, of course, would not hybridize to non-cpn60 nucleic acids under the same assay conditions.
  • 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 Laboratory Manual, 2 nd Ed., Cold Spring Harbor Laboratory Press, 1989.
  • a cpn ⁇ O nucleic acid sequence demonstrating greater than 75%, 80%, 85%, 90%, or 95% sequence identity to at least a second cpn ⁇ O nucleic acid sequence may be useful as universal cpn ⁇ O nucleic acids.
  • cpn ⁇ O coding sequences from, for example, B. anthracis, or sequences derived therefrom may cross-hybridize under the appropriate assay conditions or have sufficiently similar sequences to cpn60 coding sequences from, for example, B. anthracis and C. botulinum.
  • varying hybridization stringencies can be tested to ascertain optimal conditions for cross-hybridization. Designing such universal cpn60 nucleic acids allows for the use of a single set of reagents to detect the presence of more than one species of microbes.
  • the universality of a universal cpn60 nucleic acid can be manipulated depending on the methods used and the desired results of the assay. If, for example, the above-described universal cpn ⁇ O nucleic acids are able to detect microbes in a sample other than those used in bioterrorism, those microbes that are specifically used in bioterrorism can be specifically detected or identified using species-specific cpn ⁇ O nucleic acids. Alternatively, universal cpn ⁇ O nucleic acids can be designed to be universal with respect to only those microbes that are potentially or traditionally used in bioterrorism.
  • a cpn60 polypeptide marker is a polypeptide that includes all or a portion of a cpn ⁇ O protein. As with cpn ⁇ O nucleic acids, a cpn60 polypeptide marker can be specific to a particular species of microbe or universal to more than one species. A species-specific cpn ⁇ O polypeptide marker is.all or a portion of the cpn60 protein from a particular species of microbe, wherein detection of that species' cpn ⁇ O protein is specific over detection of cpn ⁇ O proteins from other species of microbes.
  • the probe or analytical method for detecting the marker should be capable of discriminating between the particular cpn60 polypeptide and other cpn60 polypeptides, e.g., by mass in mass-spectrometry applications or by a particular epitope in an antibody assay.
  • 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 of microbe in a sample.
  • a cpn60 polypeptide marker can be universal.
  • a "universal" cpn ⁇ O polypeptide marker may be a common structural
  • Nucleic acid-based methods for detecting the presence or absence of a microbe used in bioterrorism and, if desired, identifying and/or quantitating the amount of the 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 used to determine 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 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 microbial evaluation.
  • probes can be designed to preferentially hybridize to amplification products from individual species or to discriminate specific species.
  • PCR typically employs two 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 cpn ⁇ O 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 species of microbe such as B. anthracis, or can be designed to amplify a sequence from more than one species of microbe. 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 messenger RNA (mRNA).
  • the template nucleic acid need not be purified; it can be a minor fraction of a complex mixture, such as microbial nucleic acid contained in animal cells.
  • Template DNA or RNA can be extracted from a sample (e.g., biological or non-biological sample) using routine techniques such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al., eds., 1993, American Society 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 Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995).
  • PCR amplification product 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.
  • 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, quantitated.
  • Fluorescent analysis and quantitation 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 fiuorometer.
  • Excitation to initiate energy transfer can be carried 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.
  • corresponding refers to an acceptor fluorescent moiety having an 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.
  • Fluorescent donor and corresponding acceptor moieties are generally chosen for (a) high efficiency F ⁇ rster 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.
  • 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 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
  • acceptor fluorescent moieties depending upon the donor fluorescent moiety used, include LCTM-Red 640, LCTM-Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamme 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, hie. (Eugene, OR) or Sigma 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 nucleotide base to the fluorescent moiety.
  • A Angstroms
  • 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.
  • 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.
  • C6-Phosphoramidites available from ABI (Foster City, CA) or Glen Research (Sterling, VA)
  • 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.
  • 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)
  • 3'-amino-CPG's that require coupling of a flu
  • PCR amplification, detection, and quantitation of an amplification product can be combined in a single closed cuvette with dramatically reduced cycling time. Since detection and quantitation 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 turnaround 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 used to practice the methods of the invention.
  • a LightCyclerTM instrument is 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 is controlled by alternating heated and ambient air.
  • the cuvettes 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 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 micro volume samples.
  • 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.
  • 2 011 468) includes three band-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.
  • 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 immediately after each measurement. Fluorescent acquisition time is 10-100 milliseconds (msec). After each 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 amplification product.
  • the presence of an amplification product is indicative of the presence of one or more cp «(50-containing microbes.
  • a "cpn ⁇ O- containing microbe” refers to a microbe that contains cpn ⁇ O nucleic acid sequences.
  • the presence of FRET indicates the presence of one or more cpn ⁇ O- containing microbes in the sample, and the absence of FRET indicates the absence of ⁇ «(50-containing microbes in the sample.
  • detection of FRET within, for example, 20, 25, 30, 35, 40, or 45 cycling steps is indicative of the presence of a cp « ⁇ 50-containing microbe.
  • cpn ⁇ O 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 are designed to 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.
  • two cpn ⁇ O probes can be used, one labeled with a donor fluorophore and the other labeled with a corresponding acceptor fluorophore.
  • FRET FRET between the donor fluorescent moiety of the first cpn ⁇ O probe and the corresponding acceptor fluorescent moiety of the second cpn ⁇ O probe is detected upon hybridization of the cpn ⁇ O probes to the cpn ⁇ O amplification product.
  • a donor fluorescent moiety such as fluorescein is excited at 470 ran by the light source of the LightCyclerTM Instrument.
  • the fluorescein transfers its energy to an acceptor fluorescent 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 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 cpn ⁇ O).
  • Another FRET format can include the use of TaqMan ® technology to detect the presence or absence of a cpn ⁇ O amplification product, and hence, the presence or absence of cpra ⁇ 50-containing microbes.
  • 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. In TaqMan ® technology, the second fluorescent moiety generally is a quencher molecule.
  • the labeled hybridization probe binds to the target DNA (i.e., the cpn ⁇ O amplification product) and is degraded by the 5' to 3' exonuclease activity of the Taq Polymerase during the subsequent elongation phase.
  • the excited fluorescent moiety and the quencher moiety become spatially separated from one another.
  • the fluorescence emission from the first fluorescent moiety can be detected.
  • an ABI PRISM ® 7700 Sequence Detection System uses TaqMan ® technology, and is suitable for performing the methods described herein for detecting qwz ⁇ containing.
  • Information on PCR amplification and detection using an ABI PRISM ® 770 system can be found at the Applied Biosystems website (world wide web at appliedbiosystems.com/products).
  • Molecular beacons in conjunction with FRET also can be used to detect the presence of a cpn ⁇ O 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 formation within the probe, both fluorescent moieties are in spatial proximity when the probe is in solution. After hybridization to the target cpn ⁇ O 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.
  • 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.
  • the amount of template nucleic acid corresponds to the amount of microbe present in the sample. Therefore, the amount of FRET produced when amplifying nucleic acid obtained from a biological sample can be correlated to the amount of microbe present in a sample.
  • the amount of microbe in a sample can be quantitated by comparing the amount of FRET produced from amplification of nucleic acid in the sample to the amount of FRET produced from amplification of nucleic acid obtained from known amounts of the microbe (e.g., a standard curve). Accurate quantitation requires measuring the amount of FRET while amplification is increasing linearly.
  • the 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 microbe for comparison purposes.
  • Quantitation of microbes used in bioterrorism can be valuable prior to an attack as a method of monitoring (e.g., on a day-to-day basis) a threshold level of a microbe, while quantitation of microbes following an attack can determine the magnitude of response necessary as well as monitor the extent of exposure and the effectiveness of clean-up .
  • a cpn ⁇ O amplification product can be detected using, for example, a fluorescent DNA binding dye (e.g., SYBRGreenl ® or SYBRGold ® (Molecular Probes)).
  • a fluorescent DNA binding dye e.g., SYBRGreenl ® or SYBRGold ® (Molecular Probes)
  • DNA binding dyes 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 be used.
  • a melting curve analysis usually is performed for confirmation of the presence of the amplification product.
  • 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.
  • Tm characteristic temperature
  • 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.
  • the temperature at which signal is generated correlates with the annealing temperature of a probe with an amplification product.
  • the melting temperature(s) of cpn ⁇ O probes from an amplification product can confirm the presence or absence of cpn60- containing microbes in a sample, and can be used to quantitate the amount of a particular cpn(50-containing microbe.
  • a universal probe that hybridizes to a variable region within cpn ⁇ O will have a Tm that depends upon the sequence to which it hybridizes.
  • a universal probe may have a Tm of 70°C when hybridized to a cpn ⁇ O amplification product generated from one species of microbe (e.g., B.
  • control samples can be cycled as well.
  • Positive control samples can amplify a control nucleic acid template ⁇ e.g., a nucleic acid other than cpn ⁇ O) using, for example, control primers and control probes.
  • Positive control samples also can amplify, for example, a plasmid construct containing a cpn ⁇ O 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 cpn ⁇ O template DNA.
  • control reactions can readily determine, 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 species-specificity (if desired) and for FRET to occur.
  • methods of the invention include steps to avoid contamination.
  • an enzymatic method utilizing uracil-DNA glycosylase is 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.
  • 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.
  • FISH Fluorescent In Situ Hybridization
  • in situ hybridization methods such as FISH also can be used to detect and identify a microbe in a sample.
  • in situ hybridization methods include the steps of fixing the contents of a sample, hybridizing a cpn ⁇ O probe to DNA contained within the fixed sample, washing to remove non-specific binding, detecting the hybridized probe, and quantitating the amount of hybridized probe.
  • microbes are obtained from a sample using methods described herein. For example, microbes can be harvested by centrifuging a sample and resuspending the pelleted microbes in, for example, phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the microbes 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.
  • 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 microbes can be prepared by removing a majority of the fixative, leaving the concentrated microbes suspended in only a portion of the solution.
  • the microbe suspension is applied to slides such that the microbes do not overlap on the slide. Density can be measured by a light or phase contrast microscope. If the well containing the greatest dilution does not have enough cells, • the suspension can be concentrated and placed in another well.
  • Oligonucleotides probes for FISH are chosen for maximal sensitivity and specificity. Using a set of oligonucleotide 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 10 3 nucleotides in length (e.g., 50, 75, 100, 200, 300, 400, 500, 750, 1000, 1500, or 2000 nucleotides in length). Probes that hybridize to specific DNA can be obtained commercially from, for example, Vysis, Inc. (Downers Grove, IL), Molecular Probes, Inc. (Eugene, OR), or from Cytocell
  • oligonucleotide probes for FISH can be made non- commercially from chromosomal or genomic DNA through standard techniques.
  • sources of DNA 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 chromosomes purified by flow cytometry or microdissection.
  • the region of interest can be isolated through cloning, or by site-specific amplification via PCR.
  • Oligonucleotide probes for FISH typically are directly labeled with a fluorescent moiety (also referred to as a fluorophore), an organic molecule that fluoresces after absorbing light of lower wavelength/higher energy.
  • a fluorescent moiety also referred to as a fluorophore
  • the fluorescent moiety allows the probe to be visualized without a secondary detection molecule.
  • the nucleotide can be directly incorporated into a probe using standard techniques such as nick translation, random priming, and PCR labeling.
  • 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.
  • the number of microbes in a biological or non- biological sample can be determined.
  • fluorescent moieties of different colors can be chosen such that each probe in the set can be distinctly visualized and quantitated.
  • fluorophores 7-amino-4-methylcoumarin-3-acetic acid (AMCA), Texas RedTM (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- iso
  • AMCA 7-amino-4-methyl
  • Probes can be viewed with a fluorescence microscope and an appropriate 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 hybridization of the probes.
  • Oligonucleotide probes also can be indirectly labeled with biotin or digoxygenin, or labeled with radioactive isotopes such as P and H, although secondary detection molecules or further processing may be required to visualize the probes and quantitate the amount of hybridization.
  • 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 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.
  • the oligonucleotide probes and the chromosomal DNA contained within the microbes 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.
  • heat e.g., temperatures from about 70°C to about 95°C
  • organic solvents such as formamide and tetraalkylammonium halides, or combinations thereof.
  • 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 2X SSC (0.3 M sodium chloride and 0.03 M sodium citrate).
  • Probes can be denatured by heat (e.g., by beating to about 73°C for about five minutes).
  • Hybridizing conditions are 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.
  • in situ hybridizations typically are performed in hybridization buffer containing 1-2X SSC, 50% formamide, and blocking DNA to suppress non-specific hybridization, hi 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 oligonucleotide 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 carried 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.
  • 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.
  • assays include, without limitation, hybridization assays using detectably labeled cpn ⁇ O nucleic acid (DNA or RNA) probes and quantitative or semi-quantitative RT-PCR methodologies employing appropriate cpn ⁇ O oligonucleotide primers.
  • RNA protection assays and serial analysis of gene expression (SAGE).
  • SAGE serial analysis of gene expression
  • qualitative, quantitative, or semiquantitative in situ hybridization assays can be carried out using, for example, samples such as tissue sections or unlysed microbial suspensions, and detectably ⁇ e.g., fluorescently, isotopically, or enzymatically) -labeled DNA or RNA probes.
  • the invention also features polypeptide-based assays.
  • a cpn ⁇ O protein, or cpn ⁇ O 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).
  • the presence or absence of a cpn ⁇ O polypeptide is detected and/or its level is measured.
  • Methods of detecting or measuring the levels of a protein of interest ⁇ e.g., a cpn ⁇ O protein, or cpn ⁇ O polypeptides) in microbes are known in the art. Many such methods employ antibodies ⁇ e.g., polyclonal antibodies or mAbs) that bind specifically to the protein.
  • Antibodies having specific binding affinities for a cpn ⁇ O protein or a cpn ⁇ O polypeptide may be produced through standard methods.
  • the terms "antibody” or “antibodies” include intact molecules as well as fragments thereof which are capable of binding to an epitopic determinant of a cpn ⁇ O 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 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 “antibodies” include polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab) 2 fragments. Antibodies may be specific for a particular cpn60 polypeptide, e.g., the cpn ⁇ O protein of the B. anthracis microbe.
  • they may be cross-reactive with two or more cpn ⁇ O polypeptides, e.g., cross-react or bind to two or more cpn60 proteins.
  • such antibodies may bind to common epitopes present in two or more cpn ⁇ O proteins or cpn60 polypeptides.
  • such antibodies with specificity for two or more cpn ⁇ O polypeptides are termed "universal" antibodies.
  • certain antibodies may bind to common epitopes present in all or most cpn ⁇ O polypeptides.
  • any microbes used in bioterrorism may be present, and optionally the relative concentration or amount of such microbes.
  • detection may occur through, e.g., the use of one or more "universal" cpn ⁇ O antibodies, such as an antibody that binds or demonstrates specificity to two or more cpn ⁇ O polypeptides as described previously. Universal antibodies can be used to monitor an environment prior to an attack to determine when an attack has taken place. In other embodiments, the identification of the particular microbe may be preferred. Identification of the species of microbe used in a bioterrorism attack can determine the size of the response and the methods of containment and clean-up.
  • an antibody specific for a particular microbial species' cpn ⁇ O polypeptide may be employed; such antibodies are referred to as having binding affinity for that cpn ⁇ O polypeptide.
  • the universal and specific antibodies may be employed simultaneously or in series.
  • a universal antibody may be used as a first screen to determine the presence or absence of a cpn ⁇ O polypeptide.
  • a specific antibody such as one specific for a cpn ⁇ O polypeptide from a particular microbe, e.g., B. anthracis, may be employed.
  • monoclonal antibodies may be particularly useful (e.g., sensitive) to identify cpn ⁇ O polypeptides of a particular species of microbe.
  • a protein of interest e.g., a cpn60 protein against which one wishes to prepare antibodies
  • a protein of interest e.g., a cpn60 protein against which one wishes to prepare antibodies
  • an intact cpn60 protein may be employed, or a cpn ⁇ O polypeptide maybe employed, provided that the cpn60 polypeptide is capable of generating the desired immune response.
  • WO 02/65129 for a description of epitopic sequences from Chlamydia trachomatis that are recognized by human antibodies. See also U.S. Patent No.
  • a cpn60 polypeptide from one species of microbe may be used to generate a universal antibody, for example, if that cpn60 polypeptide maintains an epitope that is common to cpn60 proteins from at least one other species of microbe, or, e.g., to all cpn ⁇ O proteins that one wishes to detect (e.g., the cpn60 proteins from species of microbes used in bioterrorism).
  • a cpn60 protein or cpn60 polypeptide may be used to generate antibodies specific for the cpn60 protein or polypeptide from a particular microbe, e.g., only in B. anthracis.
  • Various host animals including, for example, rabbits, chickens, mice, guinea pigs, and rats, can be immunized by injection of the cpn60 protein(s) 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, pluronic polyols, polyanions, peptides, oil 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.
  • 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 et al., 1975, Nature, 256:495, the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72; Cole et al., 1983, Proc. Natl. Acad. Sd. USA, 80:2026), and the EBV- hybridoma technique (Cole et al., "Monoclonal Antibodies and Cancer TJierapy", Alan R.
  • 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 murine monoclonal antibody and a constant region derived from a human immunoglobulin. Chimeric antibodies can be produced through standard techniques.
  • Antibody fragments that have specific binding affinity for a cpn60 polypeptide can be generated by known techniques.
  • 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.
  • Fab expression libraries can be constructed. See, for example, Huse et al., 1989, Science, 246:1275.
  • Single chain Fv 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 cpn60 protein or cpn60 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, immunofluorescent assays, biosensor assays.
  • standard immunoassay methods including, for example, ELISA techniques, countercurrent immuno-electrophoresis (CIEP), radioimmunassays (RIA), radioimmunoprecipitations, dot blots, inhibition or competition assays, sandwich assays, immuno
  • Antibodies or fragments can also be tested for their ability to react universally, e.g., with cpn60 proteins or cpn60 polypeptides from more than one species of microbe, (e.g., the cpn60 proteins from B. anthracis and C. Botulinum), or specifically with a particular species' cpn60 protein (e.g., the cpn60 protein of B. anthracis).
  • the antibody itself or a secondary antibody that binds to the primary antibody can be detectably labeled.
  • the antibody can be conjugated with biotin, and detectably-labeled avidin can be used to detect the presence of the biotinylated antibody.
  • Combinations of these approaches can be used to enhance the sensitivity of assays.
  • Some of these assays e.g., immunohistological methods or fluorescence flow cytometry
  • the methods described below for detecting a cpn60 polypeptide in a liquid sample can also be used to detect a cpn60 polypeptide in lysates.
  • Methods of detecting a cpn60 polypeptide in a liquid sample generally involve contacting a sample of interest with an antibody that binds to a cpn60 polypeptide and determining whether or not the antibody bound to a component of the sample, hi such assays the antibody need not be detectably labeled and can be used without a second antibody that binds to a cpn60 polypeptide.
  • an antibody with binding affinity for a cpn60 polypeptide may be bound to an appropriate solid substrate and then exposed to the sample.
  • Binding of a cpn60 polypeptide to the antibody on the solid substrate may be detected by exploiting the phenomenon of surface plasmon resonance, wherein binding results in a change in the intensity of surface plasmon resonance that can be detected qualitatively or quantitatively by an appropriate instrument, (e.g., a Biacore apparatus (Biacore International AB, Rapsgatan, Sweden).
  • an appropriate instrument e.g., a Biacore apparatus (Biacore International AB, Rapsgatan, Sweden).
  • assays for detection of a cpn60 polypeptide in a liquid sample can involve the use, for example, of: (a) a detectably-labeled single antibody specific for a cpn60 polypeptide; (b) an unlabeled antibody that is specific for a cpn60 polypeptide and a detectably-labeled secondary antibody; or (c) a biotinylated antibody specific for a cpn60 polypeptide and detectably-labeled avidin.
  • combinations of these approaches including "multi-layer” assays) familiar to those in the art can be used to enhance the sensitivity of such assays.
  • the sample or an aliquot of the sample suspected of containing a microbe used in bioterrorism can be immobilized on a solid substrate, such as a nylon or nitrocellulose membrane, by, for example, "spotting" an aliquot of the liquid sample or by blotting of a gel on which the sample or an aliquot of the sample has been subjected to electrophoretic separation.
  • a solid substrate such as a nylon or nitrocellulose membrane
  • the presence or amount of cpn ⁇ O polypeptide on the solid substrate is then assayed using any of the above-described forms of a cpn ⁇ O polypeptide specific antibody and, where required, appropriate detectably-labeled secondary antibodies or avidin.
  • the invention also features "sandwich" assays.
  • sandwich assays instead of immobilizing samples on solid substrates by the methods described above, any cpn ⁇ O polypeptide that may be present in a sample can be immobilized on the solid substrate by conjugating a second ("capture") antibody (polyclonal or mAb) having binding affinity for a cpn ⁇ O polypeptide to the solid substrate prior to exposing the solid substrate to the sample by any of a variety of methods known in the art. Exposing a sample to the solid substrate containing the second antibody specific for cpn60 polypeptide, any cpn60 polypeptide in the sample (or sample aliquot) will bind to the second antibody on the solid substrate. The presence or amount of cpn ⁇ O polypeptide bound to the conjugated second antibody is then assayed using a "detection" antibody specific for a cpn60 polypeptide by methods essentially the same as those described above
  • 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.
  • 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 the epitope to which the capture mAb binds; or (b) a polyclonal antibody that binds to epitopes other than or in addition to that to which the capture mAb binds.
  • 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 the epitope to which the capture mAb binds; or (b) a polyclonal antibody that binds to epitopes other than or in addition to that to which the capture mAb binds.
  • the detection antibody can be either
  • a polyclonal antibody that binds to epitopes other than or in addition to that to which the capture polyclonal antibody binds include sandwich ELISAs, sandwich Western blotting assays, and sandwich immunomagnetic detection assays.
  • Suitable solid substrates to which the capture antibody can be bound include, without limitation, 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 immunoaffmity purification of cpn60 polypeptides. Dipstick/immunostick formats can employ a solid phase, e.g., polystyrene, paddle or dispstick.
  • Labels include, without limitation, radionuclides (e.g., 125 1, 131 1, 35 S, 3 H, 32 P, 33 P, or 14 C), fluorescent moieties (e.g., fluorescein, rhodamine, or phycoerythrin), luminescent moieties (e.g., QdotTM nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, CA), or enzymes (e.g., alkaline phosphatase or horseradish peroxidase).
  • radionuclides e.g., 125 1, 131 1, 35 S, 3 H, 32 P, 33 P, or 14 C
  • fluorescent moieties e.g., fluorescein, rhodamine, or phycoerythrin
  • luminescent moieties e.g., QdotTM nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, CA
  • enzymes e.g., alkaline phosphat
  • the products of reactions catalyzed by appropriate enzymes can be, without limitation, fluorescent, luminescent, or radioactive, or they may absorb visible or ultraviolet light.
  • 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.
  • concentration of a cpn60 polypeptide in, for example, a food sample suspected of being contaminated, or at risk of being contaminated, with, for example, C. hotulinum microbes that produce botulinum toxin may be compared to a control sample, e.g., a food sample known not to be infected.
  • the control sample may be taken from the same or a closely related environment, e.g., in a different location known to be uncontaminated, or can be a control sample taken from an environment completely removed from the putative bioterrorism attack (e.g., an equivalent sample from a different building).
  • control sample may be taken from the same location but at an earlier or later time-point when the location was known to be uncontaminated.
  • a significantly higher concentration of cpn ⁇ O polypeptide in the suspect sample relative to the control sample would indicate the presence of one or more microbes used in bioterrorism.
  • 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, air samples, or buffer samples ⁇ e.g., buffer used to extract a sample from a fomite).
  • the present invention also contemplates the use of other analytical techniques for detecting the presence, absence, or amount of cpn60 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, Jungblut, 2001, Microbes & Infection, 3:831-840; MacBeath and Schreiber, 2000, Science, 289:1760-1763; Madoz-Gdrpide et al., 2001, Proteomics, 1:1279-1287; Patterson, 2000, Physiological Genomics, 2:59-65; and Schevchenko et al., 2000, Analytical Chemistry, 72:2132-2141.
  • Mass-spectrophotometric techniques have been increasingly used to detect and identify proteins and protein fragments at low levels, e.g., fmol 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.
  • TOF time-of-flight
  • 0.5 ⁇ l of sample that contains 1-10 pmol of protein or peptide is mixed with an equal volume 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 ⁇ -hydroxycinnamic acid.
  • the co-crystallized material on the target plate is irradiated with a nitrogen laser pulse, e.g., at a wavelength of 337 nm, to volatilize and 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 polypeptides. See, for example, Downard, 2000, J. Mass. Spectrom. 35:493-503.
  • Biomolecular interaction analysis mass spectrometry is another suitable technique for detecting interactions between cpn60 polypeptides and cpn60 antibodies.
  • This technology detects molecules bound to a ligand that is covalently attached to a solid surface.
  • changes occur in the refractive index at the solution or surface interface.
  • This change in the refractive index is detected because the angle or wavelength at which the incident light is absorbed at the surface changes upon binding.
  • 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 plasmon resonance (SPR), as discussed previously.
  • SPR surface plasmon resonance
  • a ligand e.g., a cpn60 antibody, is covalently immobilized on the surface of a chip.
  • a tryptic digestion of solubilized proteins from a sample is routed over the chip, and the relevant peptides, e.g., cpn60 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 sub-femtomol levels.
  • Mass spectrometers useful for such applications are available from Applied Biosystems (Foster City, CA); Bruker Daltronics (Billerica, MA) and Amersham 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 subjected 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, Washburn et al., 2001, Nature Biotechnology, 19:242-247.
  • MUDPIT multidimensional protein identification technology
  • MS-MS tandem mass spectrometry
  • Articles of Manufacture can include at least one cpn ⁇ O oligonucleotide primer, as well as instructions for using the cpn ⁇ O oligonucleotide(s) to identify and quantitate the amount of one or more microbes in a sample.
  • the instructions also will relate to use of the reagents in a bioterrorism context such as a threat or actual emergency.
  • the cpn60 oligonucleotide ⁇ ) are attached to a microarray (e.g., a GeneChip ® , Affymetrix, Santa Clara, CA).
  • an article of manufacture can include one or more cpn60 oligonucleotide primers and one or more cpn ⁇ O oligonucleotide probes.
  • Such cpn ⁇ O primers and probes can be used, for example, in real-time amplification reactions to amplify and simultaneously detect cpn ⁇ O amplification products.
  • Suitable oligonucleotide primers include those that are complementary to highly conserved regions of cpn ⁇ O and that flank a variable region. Such universal cpn ⁇ O primers can be used to specifically amplify these variable regions, thereby providing a sequence with which to identify microbes used in bioterrorism. Examples of universal cpn.60 oligonucleotide primers include the following:
  • Suitable oligonucleotide primers also include those that are complementary to • species-specific cpn ⁇ O sequences, and thus result in an amplification product only if a ' ⁇ particular species of microbe is present in the sample, hi another embodiment, an article of manufacture of the invention can include a full complement of species- specific cpn ⁇ O nucleic acids to detect any microbe that is potentially or traditionally used in bioterrorism.
  • cpn ⁇ O nucleic acids that are specific to each of B. . anthracis, C. botulinum, Y. pestis, and F. tularensis can be included in an article of manufacture of the invention.
  • species-specific cpn ⁇ O nucleic acids can be used in separate but simultaneous reactions to not only detect the presence or absence of a microbe used in bioterrorism, but also to identify which microbe is present in the sample being tested.
  • cpn ⁇ O oligonucleotide probes generally are complementary to cpn ⁇ O sequences.
  • cpn ⁇ O oligonucleotide probes can be designed to hybridize universally to cpn ⁇ O sequences from more than one species of microbe, or cpn ⁇ O oligonucleotide probes can be designed for species-specific hybridization to, for example, the variable region of cpn ⁇ O nucleic acids.
  • An article of manufacture of the invention can further include additional components for carrying out amplification reactions andZor 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.
  • an article of manufacture can include one or more restriction enzymes for distinguishing amplification products from different species of microbe, or can include fluorophore-labeled oligonucleotide probes for real-time detection of amplification products.
  • an article of manufacture can be designed to detect, for example, B. anthracis.
  • a more generalized article of manufacture can be used to evaluate a number of, or all of, the microbes used in bioterrorism.
  • Articles of manufacture also or alternatively can include at least one cpn60 antibody, as well as instructions for using the same to detect the presence of a species of microbe, and optionally to identify the species of microbe in a sample, hi one embodiment, one or more cpii ⁇ O antibodies are attached to a microarray ⁇ e.g., a 96- microwell plate).
  • a microarray format may include a variety of universal and species-specific cpn ⁇ O capture antibodies; the universal and species- specific antibodies can 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 of binding of a cpn60 polypeptide to one or more capture antibodies ⁇ e.g., enzymes, substrates, buffers, and controls).
  • an article of manufacture can include one or more cpn ⁇ O antibodies attached to a dipstick.
  • dipsticks can be used, for example, to detect cpn ⁇ O polypeptides in a sample ⁇ e.g., a liquid sample).
  • Example 1 Quantitation of microbes using; universal primers and a universal probe
  • a sample is obtained from a swab of a food preparation surface in a restaurant and genomic DNA is extracted using standard methods ⁇ Diagnostic Molecular Microbiology: Principles and Applications ⁇ supra)).
  • Real-time PCR is conducted using universal cpn ⁇ O primers having the nucleotide sequences set forth in SEQ ID NO:1 and SEQ ID NO:2, and a universal cpn ⁇ O probe having the sequence 5'-GAC AAA GTC GGT AAA GAA GGC GTT ATC A-3' (SEQ ID NO:3), labeled at the 5' end with fluorescein (fluorophore; Molecular Probes, Inc.) and at the 3' end with DABCYL (quencher; (4-(4'-dimethylaminophenylazo)benzoic acid) succinimidyl ester; Molecular Probes, Inc.).
  • fluorescein fluorophore
  • DABCYL quencher; (4-(4'-dimethylaminophenylazo)benzoic acid succinimidyl ester; Molecular Probes, Inc.
  • This probe binds to a variable region of the cpn ⁇ O gene from numerous microbial species including Salmonella spp. and E. coli; 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.
  • the PCR reaction contains 3 TL extracted DNA, 1 TM each universal cpn ⁇ O primer, 340 nM universal cpn ⁇ O probe, 2.5 units Amplitaq Gold DNA polymerase (Perkin Elmer), 0.25 mM each deoxyribonucleotide, 3.5 mM MgCl 2 , 50 mM KCl, 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 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. 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' cpn ⁇ O sequence at a specific temperature, corresponding to the Tm of the universal probe and the cpn ⁇ O sequence of the particular species.
  • the step-wise loss of fluorescence at particular temperatures is used to identify the particular species or microbe present, and the loss in fluorescence of each step compared to the total amount of fluorescence correlates with the relative amount of each particular microbe.
  • a sample is obtained from food served at a National Football League (NFL) game and nucleic acids are extracted using standard methods ⁇ Diagnostic Molecular Microbiology: Principles and Applications ⁇ supra)).
  • Real-time PCR is conducted using universal cpn ⁇ O primers having the nucleotide sequences set forth in SEQ ID NO:1 and SEQ ID NO:2, and species-specific cpn ⁇ O oligonucleotide probes that hybridize to cpn ⁇ O nucleotide sequences from, and distinguish between, C. botulinum antigenic types A-G.
  • the sequences of the probes are identified by aligning cpn ⁇ O sequences from the seven antigenic types of C.
  • each antigenic type ⁇ i.e., a sequence not found in the other antigenic types.
  • Each of the species-specific probes is labeled with a different fluorescent moiety to allow differential detection of the various species.
  • the PCR reaction contains 3 TL extracted DNA, 1 TM each universal cpn ⁇ O primer, 340 nM universal cpn ⁇ O probe, 2.5 units Amplitaq Gold DNA polymerase (Perkin Elmer), 0.25 mM each deoxyribonucleotide, 3.5 mM MgCl 2 , 50 mM KCl, and 10 mM Tris-HCl (pH 8.0) in a total reaction volume of 50 TL.
  • PCR conditions include an initial incubation at 95 0 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 amount of each cpn ⁇ O amplification product.
  • the amount of each antigenic type-specific amplification product is then correlated with the amount of each antigenic type of C. botulinum by comparison to the amount of amplification product generated from samples containing nucleic acid isolated from known amounts of each antigenic type of C. botulinum.
  • a polystyrene dipstick containing two horizontal bands is constructed: one band consists of highly specific, monoclonal capture antibodies against cpn ⁇ O proteins from B. anthracis, while the other band is an internal control consisting of horseradish peroxidase.
  • a sample is collected from a filter in the ventilation system of a high-rise office building. The material collected from the filter is resuspended in PBS buffer and serial dilutions are made (1 :2, 1 :5, 1:10, etc.) directly into a detection reagent. A wetted dipstick is incubated in these dilutions for 5 minutes, and an indicator is then added to detect binding of cpn ⁇ O proteins to the capture antibodies.
  • the detection reagent includes a suitable buffer and secondary IgG detection antibodies labeled with horseradish peroxidase.
  • the indicator can be a chromogenic horseradish peroxidase substrate, such as 2,2'-AZINO-bis 3-ethylbenziazolme-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 nm.
  • the dipstick is rinsed with water (e.g., deionized water) and examined for staining of the antibody band by visual inspection. Staining of the antibody band reveals the presence of B. anthracis in the sample.
  • the internal control band provides a check on the integrity of the detection reagent.

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Abstract

Cette invention concerne des méthodes qui permettent de déterminer la présence, l'absence ou la quantité d'un microbe utilisé en bioterrorisme. Ces méthodes consistent à détecter la présence ou l'absence d'un marqueur cpn60 dans un échantillon.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1579012A2 (fr) * 2002-11-27 2005-09-28 Cargill, Incorporated Surveillance d'environnements a haut risque

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5807690A (en) * 1988-09-14 1998-09-15 Stressgen Biotechnology Corporation Method of screening physiological samples for elevated levels of heat shock proteins
US20020051985A1 (en) * 2000-05-08 2002-05-02 Whitten David G. Fluorescent polymer-QTL approach to biosensing
US20020137196A1 (en) * 2001-03-23 2002-09-26 The Regents Of The University Of California Sample preparation and detection device for infectious agents
US6803379B2 (en) * 2002-06-04 2004-10-12 Jose A. Fernandez-Pol Pharmacological agents and methods of treatment that inactivate pathogenic prokaryotic and eukaryotic cells and viruses by attacking highly conserved domains in structural metalloprotein and metalloenzyme targets

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5807690A (en) * 1988-09-14 1998-09-15 Stressgen Biotechnology Corporation Method of screening physiological samples for elevated levels of heat shock proteins
US20020051985A1 (en) * 2000-05-08 2002-05-02 Whitten David G. Fluorescent polymer-QTL approach to biosensing
US20020137196A1 (en) * 2001-03-23 2002-09-26 The Regents Of The University Of California Sample preparation and detection device for infectious agents
US6803379B2 (en) * 2002-06-04 2004-10-12 Jose A. Fernandez-Pol Pharmacological agents and methods of treatment that inactivate pathogenic prokaryotic and eukaryotic cells and viruses by attacking highly conserved domains in structural metalloprotein and metalloenzyme targets

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1579012A2 (fr) * 2002-11-27 2005-09-28 Cargill, Incorporated Surveillance d'environnements a haut risque
EP1579012A4 (fr) * 2002-11-27 2006-03-08 Cargill Inc Surveillance d'environnements a haut risque

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