WO1998020148A1 - Procede de detection de pathogenes dans des aliments - Google Patents

Procede de detection de pathogenes dans des aliments Download PDF

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Publication number
WO1998020148A1
WO1998020148A1 PCT/US1996/017535 US9617535W WO9820148A1 WO 1998020148 A1 WO1998020148 A1 WO 1998020148A1 US 9617535 W US9617535 W US 9617535W WO 9820148 A1 WO9820148 A1 WO 9820148A1
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target pathogen
sample
nucleic acid
detection
coli
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PCT/US1996/017535
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English (en)
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Prabhakara V. Choudary
Christopher M. Gooding
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The Regents Of The University Of California
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Priority to US09/308,106 priority Critical patent/US20010008887A1/en
Priority to PCT/US1996/017535 priority patent/WO1998020148A1/fr
Publication of WO1998020148A1 publication Critical patent/WO1998020148A1/fr

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    • 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
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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

Definitions

  • Salmonellosis is one of the oldest and most common food poisoning syndromes, with an overall mortality rate of up to 0.2%.
  • An estimated 4 million cases of Salmonella infection are reported each year in the US alone (Doyle, M.P. & Cliver, D.O. (1990) in Foodbome Diseases , Cliver, D.O., ed., 185-205, Academic Press, New York/ London).
  • S. typhimurium was the principal causative agent of Salmonellosis
  • S. enteritidis claim the top spot.
  • Salmonella enters the human food chain mainly through foods of animal origin, including poultry, beef, pork, cheese, mayonnaise, baby formulas and canned sea food.
  • EHEC 0157:H7 strain a prominent member of the verotoxin-producing E. coli (VTEC)
  • VTEC verotoxin-producing E. coli
  • the virulence of EHEC strains is in part due to the production of the verotoxins, shiga- like-toxin 1 (SLT-I) and shiga-like-toxin 2 (SLT-II) made up of an A (active) subunit and a B (binding) subunit (Karmali, M.A., 1989, Clinical Microbiology Reviews 2:15- 38).
  • HUS haemolytic uraemia syndrome
  • Escherichia coli O157:H7 was identified conclusively as a food pathogen in 1982, with the demonstration of its causal association with 2 outbreaks of hemorrhagic colitis, following the consumption of hamburgers (Riley, L.W., et al., 1983, New England Journal of Medicine 208:681-85; Wells, J.G., et al., 1983, Journal of Clinical Microbiology 18:512-520). Since then, it has been linked with many other outbreaks in the USA, Canada and the UK and is now the most frequently isolated diarrheagenic type of E. coli in North America (Tarr, P.I., 1995, Clinical Infectious Diseases 20:1-8). A major epidemic of E.
  • Immunoassays are more rapid and less labour-intensive than culture methods, but they are not robust enough to serve as independent procedures. Further, the interpretation of immunoassay test results is often marred by false positives, requiring further confirmation (Meng, J., et al, 1994, Trends in Food Science and Technology 5:179-185).
  • PCR polymerase chain reaction
  • VT ⁇ C VT ⁇ C
  • SLT ⁇ C an alternative nomenclature for verocytotoxic E. coli, VT ⁇ C
  • VT ⁇ C verocytotoxic E. coli
  • the invention provides a method for the specific detection of a target pathogen in a sample.
  • the method of the invention comprises, as a first step, incubating the sample under sufficient conditions to enrich the target pathogen population in the sample.
  • the target pathogen is selected by immunoselection from the sample enriched in the first step.
  • the selection of the target pathogen is followed by amplification of a nucleic acid sequence specific to the target pathogen from the target pathogen so selected.
  • the amplified nucleic acid sequence is then detected, the presence of the amplified nucleic acid sequence indicating the presence of the target pathogen.
  • the invention provides improved tools for rapid and early detection of pathogens, including E. coli O157:H7, and Salmonella strains: S. agona; S. anatum;
  • Figure 1 shows a primer set that amplifies a 215 base pair (bp) coding sequence of the A subunit of SLT-I and a 212 bp coding sequence of the A subunit of SLT-II.
  • Figure 2 shows primer sets used for PCR-detection of Salmonella.
  • Figure 3 shows the results of gel electrophoresis on samples of raw milk following a 4 hour enrichment, immunomagnetic separation and PCR amplification of fragments of the shiga-like toxin genes.
  • Figure 4 shows the results of electrophoretic detection of the SLT-gene amplification products from the immuno-PCR of ground beef samples, spiked with Escherichia coli O157:H7.
  • target pathogen means a pathogenic microorganism.
  • the target pathogen is a microbial pathogen.
  • the target pathogen is a fungus, such as yeast.
  • the target pathogen is a gram-positive bacterium. Examples of gram-positive bacteria as target pathogens include, but are not limited to, Listeria and Clostridium tyrobutyricum, the latter of which can contaminate semi-soft and hard cheeses.
  • the target pathogen is a gram-negative bacterium, such as a gram-negative enterobacterium.
  • Examples of gram-negative bacteria as target pathogens include, but are not limited to: lactic acid bacteria; Shigella; Vibrio, such as V. cholerae and V. haemolytica; Escherichia coli, such as Escherichia coli 0157:H7; and Salmonella such as S. agona; S. anatum; S. enteritidis; S. havana; S. krefeld; S. lilee; S. melegredis; S. montevideo; S. munster; S. newport; S. saintpaul; S. schwarzengrund; S. tennessee; S. typhimurium and S. worthington.
  • the target pathogen is Cryptosporidium, which can contaminate drinking water.
  • sample means any material that contains, or potentially contains, biological material which could be contaminated by the presence of a pathogenic microorganism.
  • samples for use in accordance with the invention include, but are not limited to, food samples, patient samples (e.g., feces or body fluids, such as urine, blood or cerebrospinal fluid) water, such as drinking water or other fluids.
  • Examples of a food sample include, but are not limited to: dairy products such as cheese, yogurt, ice cream or milk, including raw milk; meat such as beef, pork, minced meat, turkey, chicken or other poultry products; ground meat such as ground beef, ground turkey, ground chicken, ground pork; eggs; produce, including fruits and vegetables; peanut butter; seafood products including oysters, pickled salmon or shellfish; or juice, such as fruit or vegetable juice.
  • dairy products such as cheese, yogurt, ice cream or milk, including raw milk
  • meat such as beef, pork, minced meat, turkey, chicken or other poultry products
  • ground meat such as ground beef, ground turkey, ground chicken, ground pork
  • eggs produce, including fruits and vegetables
  • peanut butter peanut butter
  • seafood products including oysters, pickled salmon or shellfish
  • juice such as fruit or vegetable juice.
  • incubating means maintaining the sample in conditions that enhance the proliferation of the target pathogen, thereby enriching the target pathogen population in the sample.
  • the conditions that enhance the proliferation of the target pathogen will vary with the target pathogen and are known to those skilled in the art.
  • incubating is by culturing, such as culturing under standard conditions.
  • the culture media may be selective or non-selective. Examples of culture media for use in the method of the invention include, but are not limited to, pre- enrichment broth, selective enrichment broth, chloryl broth, selective agromedia or tryptic soy broth. Guidance on the selection of suitable media for different organisms may be found in AOAC International, "Food and Drug Administration Bacteriological Analytical Manual," 7th Edition, pp. 1-529, 1992.
  • selecting the target pathogen means isolating target pathogen-rich material from target pathogen-poor material, thereby obtaining a sufficiently enhanced concentration of the target pathogen so that reliable, reproducible detection of the target pathogen can be achieved.
  • selection is effected by fluorescence-activated cell sorting (facs).
  • the cells can be sorted on the basis of bioluminescence (Kricka, L.J., 1995, Analytical Chemistry 67(12):499R-502R; Tu, S.C. and Mager, H.I., 1995, Photochemistry and Photobiology 62(4):615-24; Duffy, G.
  • selection is effected by immunoselection using an antibody or fragment thereof that specifically recognizes, binds and captures the target pathogen.
  • immunoselection include, but are not limited to, selection using paramagnetic beads coated with the antibody or fragment thereof that specifically recognizes, binds and captures the target pathogen.
  • amplification of a nucleic acid sequence means any method whereby a specific nucleic acid sequence may be amplified.
  • amplification methods include, but are not limited to: polymerase chain reaction (PCR), including DNA- or RNA-based PCR, RT-PCR, immunoPCR; ligase chain reaction (LCR), self-sustained sequence replication or Q beta replicase assay.
  • the invention provides a method for the specific detection of a target pathogen in a sample.
  • the method of the invention comprises, as a first step, incubating the sample under sufficient conditions to enrich the target pathogen population in the sample.
  • the target pathogen is selected from the sample enriched in the first step.
  • the selection of the target pathogen is followed by amplification of a nucleic acid sequence specific to the target pathogen from the target pathogen so selected.
  • the amplified nucleic acid sequence is then detected, the presence of the amplified nucleic acid sequence indicating the presence of the target pathogen.
  • a single colony-forming unit (cfu) of bacterial pathogen can be detected in a contaminated food product in 8 hours or less.
  • the invention provides improved tools for rapid and early detection of pathogens, including E. coli O157:H7, and Salmonella strains: S. agona; S. anatum; S. enteritidis; S. havana; S. krefeld; S. lilee; S. melegredis; S. montevideo; S. munster; S. newport; S. saintpaul; S. schwarzengrund; S. tennessee; S. typhimurium and S. worthington, among others.
  • the invention provides a method for the detection of a target pathogen in a food sample. This method can be useful for screening food products for the presence of contaminating organisms to minimize the risk of food poisoning. In another embodiment, the invention provides a method for the detection of a target pathogen in a patient sample. This method can be useful for diagnosing infection by a microbial pathogen.
  • the incubation step of the method of the invention comprises culturing the food sample in tryptic soy broth at 37°C.
  • the conditions for optimizing the development and/or proliferation of the target pathogen will vary with the target pathogen and are known to those skilled in the art (AOAC International, Food and Drug Administration Bacteriological Analytical Manual, 7th Edition, pp. 1 - 529, 1992).
  • incubating is by culturing under standard conditions.
  • the culture media may be selective or non-selective.
  • the incubation time, temperature, media and other conditions will vary with the nature of the sample and the target pathogen, and are selected to culminate in a final titer of the target pathogen that is sufficient for subsequent detection of the amplified nucleic acid sequence.
  • One factor in determining incubation conditions is the doubling time of the target pathogen.
  • the doubling time is approximately 20 minutes.
  • an incubation of about 4 hours in tryptic soy broth at 37°C is sufficient.
  • the incubation time can be reduced to about 2 hours. The incubation time may be further reduced if a more sensitive method of detection is used.
  • Examples of more sensitive methods of detection include, but are not limited to, nucleic acid assays wherein the nucleic acid is labeled with a fluorescent intercalating dye, which allows for measurement of the amplification of nucleic acid sequences in real time (Burg, J.L. et al., 1995, Analytical Biochemistry 230(2):253-72).
  • a fluorescent intercalating reporters dye is propidium iodide.
  • nucleic acid markers examples include, but are not limited to, a chemiluminescent marker, immuno-affinity tags such as c-myc, affinity tags such as cellulose-binding domain, streptag, biotin/streptavidin or any whole or part macromolecule with a matching fit, reporter enzymes with chromogenic, luminescent, fluorescent or other tracer capabilities, or adducts that confer special and distinct properties on the target (G.H. Keller, M.M. Manak, DNA Probes, 2nd Edition, Stockton Press, New York, NY, pp. 1-659, 1993).
  • the pathogen selection step of the method of the invention comprises immunoselection using an antibody or fragment thereof that specifically recognizes, binds and captures the target pathogen.
  • immunoselection include, but are not limited to, immunomagnetic separation using paramagnetic beads coated with the antibody or fragment thereof that specifically recognizes, binds and captures the target pathogen.
  • antibodies that specifically recognize, bind and capture E. coli O157:H7 include, but are not limited to, Dynabeads anti-E. coli O157:H7 (Dynal As, Oslo, Norway).
  • a variety of antibodies for use in the method of the invention are commercially available (Accurate Chemical and Scientific Corporation, Westbury, NY; Affinity Bioreagents Inc., Golden, CO; Pierce, Rockford, IL).
  • the antibody or fragment thereof for use in the immunomagnetic separation can be attached to the paramagnetic beads according to methods known in the art (Hermanson, G.T., Bioconjugate Techniques, Academic Press, New York, NY, pp. 1-659, 1996).
  • the paramagnetic beads for use in immunoselection can comprise, for example, metal, glass, sugar, cellulose or polymer.
  • the binding affinities will vary with the material used for the beads.
  • the amplification of a nucleic acid sequence specific to the target pathogen is by polymerase chain reaction (PCR).
  • primers that can be used for amplification of nucleic acid sequences specific to E. coli O157:H7 include, but are not limited to, a primer set that amplifies a 215 base pair (bp) coding sequence of the A subunit of SLT-I and a 212 bp coding sequence of the A subunit of SLT-II, such as:
  • the invention includes methods in which other primers that are at least 80% homologous to the target nucleic acid sequence of the pathogen are used for the amplification of nucleic acid sequences specific to the target pathogen.
  • the target nucleic acid sequence of the target pathogen can be any nucleic acid sequence specific to the target pathogen, including sequences from the chromosomal/organellar genome(s), plasmid regions, lysogenic phages, genomic sequences, ribosomal genes, among others.
  • the invention also includes methods in which other primers derived from the sequences of SLT-I and SLT-II that encompass at least about a 100 bp sequence for the amplified product are used for the amplification of nucleic acid sequences specific to the target pathogen.
  • the invention also includes methods in which other primers that are derived from the sequences of other toxins produced by microbial pathogens are used for the amplification of nucleic acid sequences specific to the target pathogen.
  • the person skilled in the art can select primers for amplification of nucleic acid sequences specific to the target pathogen from the published literature or from public databases. The factors affecting the identification of suitable primers for this purpose will vary with the nature of the target pathogen.
  • primers will be selected for their specificity for the particular strain.
  • primers will be selected for their ability to amplify nucleic acid sequences of any member of the group of pathogens.
  • the skilled person can select the appropriate primers.
  • the amplification of nucleic acid sequences specific to the target pathogen is by PCR.
  • the amplification of nucleic acid sequences specific to the target pathogen is by RNA PCR.
  • the amplification is by immunoPCR.
  • the amplification is by cascade amplification.
  • the amplification is by ligase chain reaction (LCR; Wiedmann, M. et al, 1994, PCR Methods & Application 3(4):S51 -64; Zebala, J.A. and Barany, F., 1993, J. Clin. Gastroenterol. 17(2):171-5; Laffler, T.G. et al., 1993, Annales de Biologie Clinique 51(9):821-6).
  • the amplification is by new strand displacement assay.
  • the amplification is by self-sustained sequence replication.
  • the amplification is by Q beta replicase assay (Tyagi, S.
  • This lysing or detergent step will facilitate access to DNA in spores. Vegetative cells are easily lysed with a heating/microwaving procedure (G.H. Keller, M.M. Manak, DNA Probes, 2nd Edition, Stockton Press, New York, NY, pp. 1-659, 1993). No lysing or detergent step is necessary with many target pathogens, such as E. coli 0157:H7.
  • the detection of the amplification product is by gel electrophoresis, such as agarose gel electrophoresis.
  • the detection of the amplification product is by hybridization with a nucleic acid probe or probes.
  • the hybridization can be solid phase or liquid phase.
  • the hybridization can be dot hybridization with a nucleic acid probe or probes.
  • the dot hybridization can be a radioactive dot blot or non-radioactive dot blot.
  • the dot hybridization can be a dot blot micromethod or other method.
  • the detection of the amplification product is by real time fluorescence measurement.
  • the detection of the amplification product is by immunoenzymatic assay using anti-RNA:DNA hybrid antibodies.
  • the detection of the amplification product is by nucleic acid probe assay using a fluorescein dye and/or fluorescent dye photometry.
  • the detection of the amplification product is by use of immobilized nucleic acid probes.
  • substrates upon which the nucleic acid probes can be immobilized include, but are not limited to, a microplate, a membrane filter, a tube or a microchip.
  • a DNA probe is bound to a microwell plate.
  • a labeled amplification product is then allowed to bind the DNA probe.
  • the amplification product can be labeled, for example, with biotin, a colorimetric probe or a fluorescent dye.
  • the presence of amplification product can then be detected by detection of the label, or where applicable, by detection of a secondary label that binds to or reacts with the labeled amplification product, such as horseradish peroxidase, alkaline phosphatase, labeled anti-biotin antibody, avidin, avidin-biotin complex or avidin-streptavidin complex.
  • a secondary label that binds to or reacts with the labeled amplification product, such as horseradish peroxidase, alkaline phosphatase, labeled anti-biotin antibody, avidin, avidin-biotin complex or avidin-streptavidin complex.
  • the detection of the amplification product is by detection of a radioactive label associated with the nucleic acid probe.
  • methods for detecting radioactive labels associated with nucleic acid probes include, but are not limited to, autoradiography and scintillation counting (G.H. Keller, M.M. Manak, DNA Probes, 2nd Edition, Stockton Press, New York, NY, pp. 1-659, 1993).
  • the detection of the amplification product is by detection of a non-radioactive label associated with the nucleic acid probe. Examples of methods of detecting non-radioactive labels associated with nucleic acid probes include, but are not limited to, colorimetric, fluorometric or dye reporter assays.
  • the detection of the amplification product is by DNA hybridization/reverse hybridization. In another embodiment, the detection of the amplification product is by RNA synthesis. Nucleic acid probes for use in the detection of amplified nucleic acid sequences can be labeled during or following oligonucleotide synthesis.
  • the invention provides a method for the specific detection of Escherichia coli 0157:H7 in a food sample.
  • a first step of this method comprises incubating the food sample in a tryptic soy broth (TSB) at about 37°C for a sufficient time to allow for detection of subsequently amplified nucleic acid sequences. In one embodiment, this incubation is for about 2 to about 4 hours.
  • TLB tryptic soy broth
  • the food sample is contacted with magnetic beads coated with an antibody that specifically binds Escherichia coli 0157:H7.
  • the material bound to the magnetic beads is then selected from the food sample and nucleic acid sequences specific to Escherichia coli 0157:H7 are amplified by PCR.
  • the amplified nucleic acid sequences are then detected, the presence of amplified nucleic acid sequences indicating the presence of Escherichia coli 0157:H7 in the food sample.
  • the invention provides a method for the detection of
  • a first step of this method comprises incubating the food sample under sufficient conditions to culminate in a final titer suitable for subsequent detection of amplified nucleic acid sequences.
  • the food sample is contacted with magnetic beads coated with an antibody that specifically binds Salmonella.
  • the material bound to the magnetic beads is then selected from the food sample and a set of primer sequences shown in Figure 2 is used to amplify by PCR specific nucleic acid sequences recognized by these primers.
  • the amplified nucleic acid sequences are then detected, the presence of the amplified nucleic acid sequences indicating the presence of Salmonella in the food sample.
  • the primer sequences amplified by PCR are those of set #1 shown in Figure 2.
  • the primer sequences amplified by PCR are those of set #4 shown in Figure 2.
  • Methods for each step of the invention incubating samples, selecting for a target pathogen, amplifying nucleic acid sequences and detecting amplified nucleic acid sequences; are amenable to automation. Automation of any or all of the steps of the invention can further reduce the total assay time for detection of a target pathogen in a sample.
  • the method of the invention permits the detection of a target pathogen with increased speed and increased specificity. Increased speed, or reduced detection time, is achieved through the unexpected synergistic effect of combining the incubation step with both immunoselection for the target pathogen and amplification of nucleic acid sequences specific to the target pathogen. Increased specificity is achieved through the use of a primer set that is specific for the target pathogen.
  • Example 1 Detection of Escherichia coli O157:H7 in raw milk and ice cream Escherichia coli 0157: H7 (EHEC) in spiked samples of raw milk and ice cream was enriched in tryptic soy broth for 4 h, captured by immunomagnetic separation, subjected to amplification by polymerase chain reaction of parts of the verotoxin genes (SLT-I and SLT-II), and detected by agarose gel electrophoresis. Using this method, as few as one colony forming unit of E. coli 0157:H7 per g of food could be detected in less than 10 h.
  • Escherichia coli O157:H7 strains used in this study their serotype, shiga-like toxin gene status, source and the result of the polymerase chain reaction using primers to the shiga-like toxin gene are presented in Table 1.
  • E. coli 0157:H7 was grown overnight in 50 ml of tryptic soy broth (TSB; Difco) in a 250 ml Erlenmeyer flask, shaking at 120 rpm at 37°C. Ice cream (Haagen-Dazs vanilla flavour, Teaneck, NJ 07666, USA) was defrosted, aseptically unsealed and 45 g aliquots placed in 50 ml centrifuge tubes. The overnight culture was inoculated into and mixed with an ice cream sample, which was then diluted with the other ice cream samples in a ten-fold dilution series.
  • TAB tryptic soy broth
  • a decimal serial dilution of the overnight culture was made in TSB, and each dilution (1 ml) was mixed with 39 ml of raw bovine milk (UC Davis Dairy) in 50 ml centrifuge tubes. The spiked milk (200 ml) was then plated on SMACCT agar to obtain a plate count, and the remainder was stored overnight at 4°C, to simulate industrial storage and distribution practices.
  • Raw milk (10 ml) or ice cream (10 g) was aseptically removed from each spiked sample and resuspended in 90 ml of TSB in 250 ml Erlenmeyer flasks. The cultures were then incubated with shaking at 120 rpm at 37°C for 4 h.
  • PCR primers were synthesised by the UC Davis Protein Structure Laboratory, using an Applied Biosystems automatic DNA synthesiser Model 380A. This primer set amplifies a 215 base pair (bp) coding sequence of the A subunit of SLT-I and a
  • the PCR amplification mix contained a final concentration of 10 mM Tris-HCl pH 9.0, 50 mM KC1, 1 ml/1 Triton X-100, 1.8 mM MgCl 2 , 200 ⁇ M each of the deoxynucleoside triphosphates (Pharmacia, Piscataway, NJ 08855, USA), 1.5 units of Taq polymerase (Promega, Madison, WI 53711, USA) and 0.5 ⁇ M each of the primers, in a total reaction volume of 100 ⁇ l.
  • the PCR mix was added to the beads and overlaid with 70 ⁇ l of mineral oil.
  • the PCR amplifications were performed in a thermocycler (model PTC 150, M.J.
  • H ⁇ elll-digested fX174 DNA was used as a size marker.
  • the DNA bands in the gels were visualised by UV transillumination and photographed.
  • the fidelity of the primers to both forms of the SLT gene was established by testing 5 ⁇ l aliquots of the enriched cultures of all E. coli strains (Table 1) in the PCR.
  • PCR products of the expected size (212-215 bp) for the SLT genes were obtained with all the strains tested, with the exception of J5 and HF3 both of which do not carry either of the SLT genes.
  • Contamination levels of strains HFl, HF4 and HF5 ranging between zero and 10 8 cfu/g of ice cream were used in validation of the procedure. In all cases when the initial inoculum was 1 cfu/g or greater a PCR product of the expected size for SLT genes was obtained, demonstrating the high sensitivity of the method in the detection of E. coli O157:H7. The unspiked samples did not give a PCR product. Positive control PCR amplifications containing E. coli 0157: H7 strain HFl cells and negative control PCR amplifications containing no cells, consistently tested positive and negative, respectively.
  • the milk samples were then inoculated with serial dilutions of E. coli O157:H7 and stored at 4°C for at least 16 h, to cold-stress the bacteria. Plate counts of E. coli O157:H7 in the milk samples on SMAC agar before and after the cold-treatment showed no measurable reduction indicating that the storage conditions had no effect on E. coli O157:H7 viability.
  • the results of a validation experiment using strain HFl are presented in Fig 1. With contamination levels ranging from 4 to 3x10 5 cfu/ml of milk, all the spiked samples tested PCR-positive, but none of the unspiked. Further experiments using strains HFl, HF52, HF4 and HF5 at 1 cfu/ml or greater consistently gave SLT-positive PCR products.
  • PCR polymerase chain reaction
  • cfu colony forming units
  • Figure 3 shows the results of gel electrophoresis of samples following the 4 hour enrichment followed by immunomagnetic separation to remove E. coli 0157:H7 cells and PCR amplification of fragments of the shiga-like toxin genes.
  • Lane 1 shows unspiked sample; lane 2, sample spiked with 4 cfu/ml; lane 3, sample spiked with 36 cfu/ml; lane 4, sample spiked with 4 x 103 cfu/ml; lane 5, sample spiked with 3 x 105 cfu/ml; lane 6, negative control, no DNA or cells added; lane 7, positive control containing strain HFl cells.
  • Lane M is ⁇ X174 DNA marker digested with Haelll (the 234 bp and 194 bp markers are noted to the right).
  • Enriching in non-selective media increases the likelihood of detecting bacteria that have been stressed chemically, mechanically, or by temperature during manufacturing processes.
  • a selective agent such as novobiocin, cefimide or potassium tellurite is included in the enrichment broth to reduce the numbers of competing bacteria and to allow enrichment of the target bacteria.
  • novobiocin in the enrichment broth prevented the growth of E. coli 0157: H7 when the cells had been cold-stressed (4°C for 4 days in TSB).
  • Our observation corroborates the finding of Mackey (Mackay, 1988, FEMS Microbiology Letters 20:395-399) that various selective agents, including novobiocin, reduce the viability of E. coli cells subjected to physical stress.
  • the benefits of using a non-selective enrichment media outweighed those of using a selective media.
  • IMS In food-related applications, IMS is predominantly used for the separation of microbial cells from the food matrix and non-target microbes (Okrend, A.J.G., Rose, B. ⁇ . & Lattuada, C.P., 1992, Journal of Food Protection 55:214-217; Wright, D. J., Chapman, P. A. & Siddons, C. A., 1994, Epidemiology and Infection 113:31-39).
  • Microscopic paramagnetic particles coated with pathogen-specific antibodies concentrate the target cells from the enrichment broth. We used magnetic beads in our procedure as a selective enrichment step to capture E. coli 0157: H7.
  • the IMS step in addition to selectively concentrating the target microbe from a heterogeneous microbial population present in the test food samples, also reduces potential interference from food ingredients by separating the bacteria from the food homogenate. This is important because although PCR is a robust method, there have been reports of food components interfering with the PCR (Powell, H. A., et al., 1994, Letters in Applied Microbiology 18:59-61). Our results, using IMS, showed no evidence for PCR inhibition by the food matrix. However, we observed that the capture of the E. coli onto the immunomagnetic beads was not 100% efficient because up to 25% of them were lost during the washing step.
  • PCR product of the correct size serves as a quick and definitive confirmation of the identity of the target organism.
  • the fidelity and target-specificity of the primers determine the confidence level of the PCR procedure for the confirmation of the pathogen identity.
  • a number of primer sets with specificity to EHEC e.g., those based on the SLT-I and SLT-II genes, have been designed and successfully used by others to detect EHEC from a variety of matrices, e.g., faeces, ground beef (Paton, et al.., 1993, Journal of Clinical Microbiology 31 :3063-3067; Gannon, V. P.
  • E. coli O157:H7 cells were separated quickly from the enrichment culture, using magnetic beads coated with E. coli O157:H7-specific antibody; the bead-bound bacterial cells were subjected to polymerase chain reaction (PCR) to amplify specific segments of the SLT-I and SLT-II genes of E. coli O157:H7, and the amplified DNA was detected by agarose gel electrophoresis. Detection of a single colony forming unit (cfu) of E. coli O157:H7 was achieved in 8 h. Strains
  • Table 3 lists the bacterial strains used in the study. Counts of E. coli 0157: H7 were obtained by plating on SMACCT (Zadik, P.M., Chapman, P.A. & Siddons, C.A., 1993, Journal of Medical Microbiology. 39:155-158)) agar plates.
  • Table 3 Bacterial strains used. SLT gene status and sources
  • the frozen meatballs were each transferred to the mesh insert of a stomacher bag (Applied Biosolutions, New York), and were thawed at room temperature for 30 min. Then, 225 ml of TSB were added to each bag, and the meatballs were homogenized for 1 min in a Stomacher (Col worth). The meat homogenate was then incubated in the bag at 37°C for 4 h.
  • the PCR amplifications were carried out as described in Example 1 , using a primer set derived from the Shigella-like toxins, SLT-I and SLT-II.
  • the primers 5'- ATACAGAG(GA)G(GA)ATTTCGT-3' and 5'-TGATGATG(AG)CAATTCAGTAT- 3', amplify the 215-bp and the 212-bp segments encompassing the 586-800 bp of the SLT-I coding sequence and the 583-794 bp of the SLT-II coding sequences, respectively.
  • Paton et al. (1993, Journal of Clinical Microbiology 31 :3063-3067) designed these primer sequences and demonstrated their E.
  • the PCR buffers 100 ⁇ l reaction volumes) containing final concentrations of: 10 mM Tris-HCl, pH 9.0, 50 mM KC1, 1.8 mM MgCl 2 , 1 ml/ L Triton X-100, 200 ⁇ M each of the deoxynucleoside triphosphates (Pharmacia), 1.5 U of Taq polymerase (Promega) and 0.5 ⁇ M each of the primers, were added to the immunomagnetic beads (20 ul), mixed and then transferred to a PCR tube and overlaid with 70 ⁇ l of mineral oil.
  • the amplifications were performed in a thermocycler (M.J. Research, model PTC150) with a 2-minute initial denaturation at 94°C, followed by 35 cycles of denaturation at 92°C for 1 min, primer annealing at 48°C for 1 min and extension at 72°C for 1 min.
  • the products (10 ⁇ l) from each reaction were resolved by electrophoresis for 30 min at a constant power of 100 V, in a 1.5% agarose gel in Tris- actate-EDTA (TAE) buffer, both containing 0.5 ⁇ g/ml ethidium bromide.
  • TAE Tris- actate-EDTA
  • Hae ⁇ il digest of fX 174 DNA was used as DNA size markers. The DNA bands in the gels were illuminated by UV light and were photographed.
  • the principal goal of our method is to reduce the overall detection time, without compromising the specificity or sensitivity.
  • the total assay time can be considerably reduced when these steps are used together rather than individually. Accordingly, we first optimized each of these steps and then integrated them into a single method.
  • the pre-enrichment step helps by increasing the bacterial cell number to the minimum detection threshold.
  • this step has been carried out by others for considerably long periods, ranging from 6 to 48 h, using selective media that promote the growth of the target pathogen and suppress the growth of non-target organisms.
  • such media can impede the recovery of stressed organisms, including the target organism.
  • the use of a non-selective enrichment allowed us to enrich E. coli 0157:H7 (with a doubling time of 26 min) within 4 h from a single cfu to 500 cfu, the minimal number of target bacterial cells needed for ensuring the specificity and sensitivity of the subsequent PCR step. It did not appear that the growth of E. coli 0157:H7 was affected by competing organisms, possibly due to the short enrichment time used at this stage.
  • primers The specificity of the primers was ascertained by testing them against a panel of target and non-target bacterial strains (Table 3). PCR products of the expected size were only obtained with E. coli strains of the O157:H7 serotype, carrying either copy of the SLT genes (I or II), but not with E. coli of any other serotype nor with different species of the non-E. coli bacteria tested (Fig. 1).
  • Lane M DNA size markers: fX 174 Hae III digest
  • Lanes 2-8 samples: lane 2, unspiked; lane 3 spiked with 2 colony forming units (cfu) /g; lane 4, spiked with 30 cfu/g; lane 5, spiked with 400 cfu/ml; lane 6 spiked with 3 x 10 4 cfu/g; lane 7, negative control, no DNA or cells added; lane 8; positive control, containing HFl cells.
  • Table 4 shows the effect of the decreasing levels of HF2, HF4 and HFl 4 on the sensitivity of the new method. Detection of 1 cfu of E. coli O157:H7 per g of meat was achieved in all the cases, using the protocol we have devised, regardless of the strain variations. One sample of beef, however, tested positive by PCR, even without spiking, indicating either a possible prior contamination of the meat at one of the source points or that the primers were detecting cross-reacting species of bacteria.
  • the experimental conditions that may need target-specific but standard alterations for its application for detection of a new target pathogen include: the template sequence, the primers, optimal PCR conditions for target sequence amplification, antibody reagent(s) when the IMS step is included, and media and culture conditions if the enrichment step is used.
  • Salmonella strains S. agona; S. anatum; S. enter itidis; S. havana; S. krefeld; S. lilee; S. melegredis; S. montevideo; S. munster; S. newport; S. saintpaul; S. schwarzengrund; S. tennessee; S. typhimurium and S. worthington, and 13 non- Salmonella strains: Aeromonas hydrophila; Bacillus cereus; Citrobacter freundii;
  • Enterobacter cloacae Enterobacter cloacae; Enter o. faecalis; E. coli non-VTEC; Hafnia alvei; Klebsiella pneumoniae; Proteus vulgaris; Pseudomonas aeroginosa; Shigella sonnei;
  • the PCR product was analyzed as a function of the band position (as an indication of the amplified DNA fragment size) on an agarose gel, and its intensity as well as its production as single or multiple
  • the PCR mixes were made up using identical concentrations of primers, dNTPs and MgCl 2 as specified in the corresponding papers, and the Taq polymerase and the buffer obtained from Promega.
  • the primer sets consisted of:
  • thermocycler M.J. Research, model PTC 150
  • TSB TSB
  • the accuracy of the temperature profiling of the thermocycler had previously been confirmed using an independent thermocouple linked to a digital thermometer.
  • the reactions (10 ⁇ l of each reaction) were analyzed by electrophoresis for 30 min at 100 V in agarose (1.5%) gel in Tris-acetate-EDTA (TAE) buffer, both containing 0.5 ⁇ g/ml ethidium bromide. H ⁇ elll-digested fX174 DNA was used as size marker. The DNA bands in the gels were visualized by UV transillumination and photographed.
  • Table 5 Summary of PCR primers, their properties and results of use in PCR- detection of Salmonella
  • Primer set #2 failed to yield PCR product with 2 different strains of S. enteritidis, although all the rest of the primer sets elicited PCR products of the expected size with these strains.
  • Primer set #5 produced multiple non-specific bands of non-target size, in addition to the target product even with Salmonella strains, i.e., S. lilee and S. melegredis. This inconsistency decreases the level of confidence of detection. Thus, of all the primers we tested, set #4 consistently showed reliable sensitivity, reproducibility and specificity.
  • Primer set #1 also appears to be amenable to optimization and use with reliable results.
  • the above discrepancies at least to an extent, may be attributed to limitations inherent to the PCR technique, including variations caused by the polymerase and the buffer, the make and model of the PCR equipment.
  • Another likely source of bias may be our direct use of whole bacteria as the template source in variance to the use of isolated DNA by the previous authors.
  • PCR products obtained with the 5 different primer sets with 13 non- Salmonella bacterial strains f, faint band; m, multiple PCR bands including Salmonella-positive sized band; n, PCR band/bands of non-target size; o, no PCR band; s, single Salmonella-positive PCR band; -, not tested.

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Abstract

Cette invention concerne un procédé permettant de détecter spécifiquement des pathogènes dans un échantillon. Dans une première étape le procédé de cette invention comprend la mise en incubation de l'échantillon dans des conditions suffisantes pour enrichir la population de pathogènes cibles présente dans l'échantillon; dans une deuxième étape le pathogène cible est sélectionné par immunosélection dans l'échantillon enrichi dans la première étape. La sélection du pathogène cible est suivie par l'amplification d'une séquence d'acide nucléiques spécifique contre le pathogène cible issu du pathogène cible ainsi sélectionné. La séquence d'acides nucléiques amplifiée est ensuite détectée, la présence de la séquence d'acides nucléiques amplifiée indiquant la présence du pathogène cible. A l'aide du procédé de cette invention une unité colonigène unique de pathogène bactérien peut être détectée dans un produit alimentaire contaminé en huit heures ou moins. Cette invention fournit des outils de détection rapide et précoce de pathogène tels que E. coli O157:H7, et les souches de Salmonella: S. agona; S. anatum; S. enteritidis; S. havana; S. krefeld; S. lilee; S. melegridis; S. montevideo; S. munster, S. newport; S. saintpaul; S. schwarzengrund; S. tennessee; S. typhimurium et S. worthington parmi d'autres. Dans une forme de réalisation cette invention concerne un procédé de détection d'un pathogène cible dans un échantillon d'aliment. Ce procédé peut être utile pour rechercher systématiquement dans des produits alimentaires la présence d'organismes contaminants afin de réduire au maximum le risque d'empoisonnement alimentaire. Dans un autre forme de réalisation cette invention concerne un procédé de détection d'un pathogène cible dans un échantillon de patient. Ce procédé peut être utile pour diagnostiquer l'infection provoquée par un pathogène microbien.
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EP0976837A2 (fr) * 1998-05-29 2000-02-02 Shimadzu Corporation Oligonucléotides pour la détection des E.coli enterales et hémorragiques et procédé de détection utilisant ces oligonucléotides
WO2000077247A1 (fr) * 1999-06-15 2000-12-21 University Of Guelph Marqueur specifique des serotypes o157:h7; o157:nm et o55:h7 de escherichia coli
WO2001031056A2 (fr) * 1999-10-27 2001-05-03 Universite De Liege Methode de detection par pcr
FR2801677A1 (fr) * 1999-11-30 2001-06-01 Ulp Ct D Analyses Et De Rech S Procede d'analyse d'un echantillon pour la presence eventuelle de legionelles comprenant une etape d'immunocapture
WO2002046464A2 (fr) * 2000-12-05 2002-06-13 Norchip A/S Procede de detection de ligands
WO2003080865A1 (fr) * 2002-03-26 2003-10-02 Council Of Scientific And Industrial Research Amorces pour detecter des bacteries qui contaminent des aliments, et leur utilisation
US7241566B2 (en) 2001-06-22 2007-07-10 Marshfield Clinic Methods and oligonucleotides for the detection of Salmonella sp., E. coli O157:H7, and Listeria monocytogenes
WO2009064766A1 (fr) 2007-11-16 2009-05-22 E. I. Du Pont De Nemours And Company Procédé de détection et/ou analyse de levures et de moisissures dans des liquides filtrables
EP2561360A1 (fr) * 2010-04-21 2013-02-27 NanoMR, Inc. Isolement de substance à analyser cible provenant de liquide biologique
WO2016079304A1 (fr) * 2015-02-17 2016-05-26 Danmarks Tekniske Universitet Procédé rapide de détection de salmonella dans la viande
US9476812B2 (en) 2010-04-21 2016-10-25 Dna Electronics, Inc. Methods for isolating a target analyte from a heterogeneous sample
US9551704B2 (en) 2012-12-19 2017-01-24 Dna Electronics, Inc. Target detection
US9599610B2 (en) 2012-12-19 2017-03-21 Dnae Group Holdings Limited Target capture system
US9696302B2 (en) 2010-04-21 2017-07-04 Dnae Group Holdings Limited Methods for isolating a target analyte from a heterogeneous sample
US9804069B2 (en) 2012-12-19 2017-10-31 Dnae Group Holdings Limited Methods for degrading nucleic acid
US9902949B2 (en) 2012-12-19 2018-02-27 Dnae Group Holdings Limited Methods for universal target capture
US9995742B2 (en) 2012-12-19 2018-06-12 Dnae Group Holdings Limited Sample entry
US10000557B2 (en) 2012-12-19 2018-06-19 Dnae Group Holdings Limited Methods for raising antibodies
WO2020106897A1 (fr) * 2018-11-20 2020-05-28 Alex Brandt Procédés et matériaux permettant de détecter la salmonella dans le bœuf
KR20200070792A (ko) * 2018-12-10 2020-06-18 대한민국(농촌진흥청장) 면역 자성 입자를 이용한 식중독균 동시검출용 조성물 및 방법

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EP0976837A2 (fr) * 1998-05-29 2000-02-02 Shimadzu Corporation Oligonucléotides pour la détection des E.coli enterales et hémorragiques et procédé de détection utilisant ces oligonucléotides
EP0976837A3 (fr) * 1998-05-29 2003-05-21 Shimadzu Corporation Oligonucléotides pour la détection des E.coli enterales et hémorragiques et procédé de détection utilisant ces oligonucléotides
WO2000077247A1 (fr) * 1999-06-15 2000-12-21 University Of Guelph Marqueur specifique des serotypes o157:h7; o157:nm et o55:h7 de escherichia coli
WO2001031056A2 (fr) * 1999-10-27 2001-05-03 Universite De Liege Methode de detection par pcr
WO2001031056A3 (fr) * 1999-10-27 2002-06-27 Univ Liege Methode de detection par pcr
FR2801677A1 (fr) * 1999-11-30 2001-06-01 Ulp Ct D Analyses Et De Rech S Procede d'analyse d'un echantillon pour la presence eventuelle de legionelles comprenant une etape d'immunocapture
WO2001040505A1 (fr) * 1999-11-30 2001-06-07 Ulp-Centre D'analyses Et De Recherches Procede d'analyse d'un echantillon pour la presence de bacteries legionella comprenant une etape d'immunocapture
WO2002046464A2 (fr) * 2000-12-05 2002-06-13 Norchip A/S Procede de detection de ligands
WO2002046464A3 (fr) * 2000-12-05 2003-09-12 Norchip As Procede de detection de ligands
US7241566B2 (en) 2001-06-22 2007-07-10 Marshfield Clinic Methods and oligonucleotides for the detection of Salmonella sp., E. coli O157:H7, and Listeria monocytogenes
WO2003080865A1 (fr) * 2002-03-26 2003-10-02 Council Of Scientific And Industrial Research Amorces pour detecter des bacteries qui contaminent des aliments, et leur utilisation
WO2009064766A1 (fr) 2007-11-16 2009-05-22 E. I. Du Pont De Nemours And Company Procédé de détection et/ou analyse de levures et de moisissures dans des liquides filtrables
EP2561360A1 (fr) * 2010-04-21 2013-02-27 NanoMR, Inc. Isolement de substance à analyser cible provenant de liquide biologique
US9476812B2 (en) 2010-04-21 2016-10-25 Dna Electronics, Inc. Methods for isolating a target analyte from a heterogeneous sample
EP2561361A1 (fr) * 2010-04-21 2013-02-27 NanoMR, Inc. Compositions et procédés pour l'isolement de bactéries en milieu de phase logarithmique
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US11448646B2 (en) 2010-04-21 2022-09-20 Dnae Group Holdings Limited Isolating a target analyte from a body fluid
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US9551704B2 (en) 2012-12-19 2017-01-24 Dna Electronics, Inc. Target detection
US10745763B2 (en) 2012-12-19 2020-08-18 Dnae Group Holdings Limited Target capture system
US9804069B2 (en) 2012-12-19 2017-10-31 Dnae Group Holdings Limited Methods for degrading nucleic acid
US9995742B2 (en) 2012-12-19 2018-06-12 Dnae Group Holdings Limited Sample entry
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WO2016079304A1 (fr) * 2015-02-17 2016-05-26 Danmarks Tekniske Universitet Procédé rapide de détection de salmonella dans la viande
WO2020106897A1 (fr) * 2018-11-20 2020-05-28 Alex Brandt Procédés et matériaux permettant de détecter la salmonella dans le bœuf
KR102145618B1 (ko) * 2018-12-10 2020-08-20 대한민국 면역 자성 입자를 이용한 식중독균 동시검출용 조성물 및 방법
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