METHOD FOR CONFIRMING THE PRESENCE OF AN ANALYTE

Abstract

The invention provides methods and kits for the rapid confirmation of an initial analyte test result. In a preferred embodiment, the process confirms the presence of a given microbial target in a mixed culture, or a mixed enrichment media, even when the competing organisms in the mix belong to related species, or are various biotypes of the same species.

Claims

1. A method of confirming the presence of a target contaminant microorganism or of a false positive in a sample, comprising: contacting a first portion of a sample that may be contaminated by a plurality of genetically distinct microorganism with a surface coated with an antibody that specifically recognizes a target contaminant microorganism to provide an antibody-captured first sample portion; providing a second portion of the sample that has not been contacted with the surface coated with the antibody; assaying the antibody-captured first sample portion for the presence of the target contaminant microorganism by using a multiplex PCR assay using PCR primers complementary to nucleic acid targets indicative of the presence of the target contaminant microorganism, and wherein at least one of the nucleic acid targets also detects at least one other genetically distinct non-target microorganism; separately assaying the second sample portion for the presence of the contaminant microorganism by using the multiplex PCR assay; and differentially comparing the results of the PCR assays wherein: a) detection of the nucleic acid targets in the multiplex PCR assay conducted on the antibody-captured first sample portion is indicative of the presence of the target contaminant microorganism in the sample, or b) detection of none or a subset of the nucleic acid targets in the multiplex PCR assay conducted on the antibody-captured first sample portion, and detection of the nucleic acid targets in the multiplex PCR assay conducted on the second sample portion is indicative of a composite, false positive, signal from the plurality of genetically distinct microorganisms and the absence of the target contaminant microorganism, wherein a method of confirming the presence of a target contaminant microorganism or of a false positive in the sample is afforded.

2. The method of claim 1, wherein prior to contacting the first sample portion with the antibody-coated surface, the sample is contacted with a growth media to enrich the sample in the target contaminant microorganism present in the sample.

3. The method of claim 1, wherein the sample has been analyzed for the presence of the target contaminant microorganism by 4-band multiplex PCR assay prior to contacting a first sample portion with the antibody-coated surface.

4-6. (canceled)

7. The method of claim 1, wherein the sample is a sample presumptively positive for the target contaminant microorganism, based on a prior test of the sample.

8. The method of claim 1, wherein assaying the antibody-captured first sample portion and separately assaying the second sample portion comprises use of a 4-band multiplex PCR assay using three PCR primer pairs complementary to three nucleic acid targets indicative of the presence of the target contaminant microorganism, and wherein one of the PCR primer pairs detects the target contaminant microorganism and the at least one other genetically distinct non-target microorganism, and wherein: (a) detection of the three nucleic acid targets indicative of the target contaminant microorganism in the 4-band multiplex PCR assays conducted on the antibody-captured first sample portion is indicative of the presence of the target contaminant microorganism in the sample, or (b) detection of the one nucleic acid target present on both the target contaminant microorganism and the at least one other genetically distinct non-target microorganism in the 4-band multiplex PCR assay conducted on the antibody-captured first sample portion, and the detection of the three nucleic acid targets indicative of the target contaminant microorganism in the 4-band multiplex PCR assay conducted on the second sample portion is indicative of a composite signal from the plurality of genetically distinct microorganisms and the absence of the target contaminant microorganism.

9. The method of claim 8, further comprising: providing to the 4-band multiplex PCR, an additional primer pair complementary to an additional nucleic acid target specific to the target contaminant microorganism so as to create a 5-band multiplex PCR assay; wherein said separately assaying the antibody-captured first sample portion for the presence of the target contaminant microorganism uses the 5-band multiplex PCR assay; wherein said separately assaying the second sample portion for the presence of the target contaminant microorganism uses the 5-band multiplex PCR assay; and differentially comparing the results of the PCR assays wherein: (a) detection of the four nucleic acid targets specific to the target contaminant microorganism in the 5-band multiplex PCR assay conducted on the antibody-captured first sample portion is indicative of the presence of the target contaminant microorganism in the sample, or (b) detection of none or a subset of the four nucleic acid targets specific to the target contaminant microorganism in the 5-band multiplex PCR assay conducted on the antibody-captured first sample portion, and the detection of all of the nucleic acid targets in the 5-band multiplex PCR assay conducted on the second sample portion is indicative of a composite signal from the plurality of genetically distinct microorganisms and the absence of the target contaminant microorganism.

10. The method of claim 1, wherein the target contaminant microorganism is selected from the group consisting of Escherichia coli 0157:H7 (E. coli 0157:H7), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterpathogenic Escherichia coli (EPEC), Salmonella, Listeria, Yersinis, Campylobacter, Clostridial species, Staphylococcus spp., frank and opportunistic bacterial, fungal, viral, parasitic pathogens; indicator organisms including heterotrophes, generic E. coli, total and fecal coliforms and enterococcus; spoilage organisms including Pseudomonas; Staph. aureus, Bacillus cereus, Clostridium botulinum, Clostridium perfringes, Vibrio cholerae and V. parahemolyticus, Yersinia enterocolitica, Yersinia pestis, Brucella, Francisella, Aeromonas, Plesiomonas, Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus, Providencia, Serratia, Shigella, and combinations thereof.

11. The method of claim 10, wherein the target contaminant microorganism is selected from the group consisting of Escherichia coli 0157:H7 (E. coli 0157:H7), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterpathogenic Escherichia coli (EPEC), Salmonella, Listeria, Yersinis, Campylobacter, Clostridial species, and Staphylococcus spp.

12. The method of claim 11, wherein the target contaminant microorganism is Escherichia coli 0157:H7 (E. coli 0157:H7).

13. The method of claim 1, wherein the antibody-coated surface comprises a magnetic bead coated with the antibody.

14. The method of claim 3, wherein the antibody-coated surface comprises a magnetic bead coated with the antibody.

15. The method of claim 8, wherein the antibody-coated surface comprises a magnetic bead coated with the antibody.

16. The method of claim 9, wherein the antibody-coated surface comprises a magnetic bead coated with the antibody.

17. The method of claim 3, wherein the target contaminant microorganism comprises E. coli O157, and the 4-band multiplex PCR assay comprises primer sets for rfb, eae, stx1, and stx2.

18. The method of claim 8, wherein the target contaminant microorganism comprises E. coli O157, and the 4-band multiplex PCR assay comprises primer sets for rfb, eae, stx1, and stx2.

19. The method of claim 18, wherein the three PCR primer pairs are complementary to rfb, eae, and stx1 or stx2, and wherein the one PCR primer pair is complementary to rfb.

20. The method of claim 15, wherein the target contaminant microorganism comprises E. coli O157, and the 4-band multiplex PCR assay comprises primer sets for rfb, eae, stx1, and stx2.

Description

DESCRIPTION OF EMBODIMENTS

[0024] The present invention is drawn to methods of screening and monitoring for microbial growth and contaminants including the use of secondary analysis methods needed for rapid screening and verification of primary or preliminary testing results. In conducting testing for microbial growth or contamination, an initial or preliminary testing result may be obtained by sampling and/or testing a subject or good by any one of several testing methodologies as described in U.S. Patent Publication No. 2006/0115824, such as presence/absence tests or a plurality of the same.

[0025] Processes and systems to which the testing and verification methods of the instant application may be applicable include, but are not limited to: food production; manufacturing; processing; storage; transportation and distribution; with respect to microbial pathogens-process sanitation, environmental contaminants, and spoilage organisms; with respect to fermentation processesdetermining purity of the seed stock and fermentation contaminants; aseptic processing (e.g., food and pharmaceutical; with respect to sterility and environmental control); water treatment (e.g., with respect to microbiological quality of the raw and treated water, and control of the organisms throughout the distribution system); wastewater treatment (e.g., with respect to microbiological quality of the treated wastewater and biosolids, control of the treatment process, control of the aerobic and anaerobic digesters, and assessment of the impact of the discharged wastewater and application of bio-solids on the receiving environments); control of microbial contaminants and assessment of their impact in the indoor environment and indoor air quality assessment studies; environmental microbiology (e.g., with respect to monitoring the microbiological quality of shellfish, shellfish beds, and cultured aquatic organisms, assessing the microbiological quality of recreational waters and swimming beaches, assessing the microbiological quality of bodies of water, conducting impact assessment of point and non-point-sources); feed microbiology (e.g., in determining the microbiological quality and safety of the feed); soil microbiology (e.g., in assessing the overall microbiology and population structure of soil organisms, in assessing target organisms that can indicate environmental contamination or organic and inorganic reservoirs (e.g., oil fields)).

[0026] If a presumptive positive result is obtained using the rapid screening method, molecular confirmation is conducted using test methods that may include, but are not limited to, multiplex PCR reaction(s), DNA chips, dot blots, multi- and single-target lateral flow devices, and combinations thereof. In preferred aspects, assays suitable for detection of pathogenic or microbial contamination may include the use of immunoassays, nucleic acid amplification-based assays, PCR-based assays, nucleic acid hybridization-based assays, bio-sensor assays, immunostaining-microscopy-based assays, nucleic acid-array-based assays, DNA chip-based assays, bacteriophage-detection-based assays, classical microbiology-based assays, and chemical or biochemical assays based on the detection of compounds associated with particular target organisms or groups of target organisms, and combinations thereof.

[0027] In a specific application for the target analyte organism E. coli O157, a 4-band multiplex PCR assay is used as an initial, rapid screening method. The four PCR assay targets are gene segments known to be associated with E. coli O157. These assay targets are rfb, eae, stx1, and stx2. After enrichment of a sample to allow the growth of the target organisms to the detection level, the enrichment is screened with the 4-band multiplex PCR assay.

[0028] If a presumptive positive result is obtained using the rapid screening method, molecular confirmation is conducted using two other multiplex PCR reactions (one with four targets and one with five targets). Each PCR assay contains targets for stx1, stx2, rfb, and eae (one universal one gamma). One of the multiplex PCR assays also contains an additional gene unique to O157. Molecular Confirmation is done using the two multiplexes, with and without immunomagnetic separation using magnetic beads coated with anti-O157 antibodies.

[0029] The key to separation of O157 from non-O157 STEC and determination of whether there is a true O157 versus a composite signal, is determined by a comparison of the PCR assays with and without the beads and the fact that 0157 carries gamma eae. If a four band multiplex assay with the magnetic beads shows rfb, eae, and six, this indicates that a true pathogenic 0157 E. coli in the enrichment, however if it only shows rfb, while the non-magnetic separated enrichment shows the rfb, eae, and six, then we know that there is a non-toxigenic, eae-negative O157 in the enrichment.

[0030] The same principle is used for Listeria monocytogenes vs. Listeria species in the same enrichment and for toxigenic Bacillus cereus versus other Bacilli in the same enrichment, and for Salmonella/multi-drug resistant Salmonella versus a multi-drug resistant bacteria in the same enrichment. The same assay principles are used for other pathogenic, spoilage, indicators, biodegridative organisms, and pharmaceutical platform/producer strains of organisms.

[0031] A fifth specific application again targets Salmonella spp. Immunomagnetic Separation (IMS) is used to purify and concentrate the sample prior to conducting a confirmation 3-band multiplex PCR assay; however, the immunomagnetic beads in this case are coated with antibodies which bind specifically to Salmonella spp.

[0032] In a sixth specific application, the pathogenic microbes Listeria spp. and Listeria monoctogenes (LM) are targeted using a 4-band multiplex PCR assay as a rapid screening method. Two of the PCR assay targets are gene segments known to be associated with LM, while the remaining two are known to be associated with Listeria spp. If a presumptive positive result is obtained using the rapid screening method for LM, a second 2-band multiplex PCR assay is used to corroborate the presumptive positive result and provide confirmation. If a presumptive positive result is obtained for Listeria spp. another independent 2-band multiplex PCR assay is used to corroborate the presumptive positive result and provide confirmation.

[0033] A seventh specific application again targets Listeria spp. and Listeria monocytogenes. IMS is used to purify and concentrate the sample prior to applying one or both of the confirmation 2-band multiplex PCR assays; however, the immunomagnetic beads in this case are coated with antibodies, which bind specifically to Listeria spp., of which LM is one type.

[0034] Another aspect of the present invention is directed to a rapid confirmation method for microorganisms, in mixed cultures or enrichment cultures. The method allows for rapid confirmation of the presence of single or multiple target organisms in the same mixture. The method has built in redundancy that increases the confidence in the results. It can also confirms and characterizes the organisms in a single step.

[0035] These microorganisms may include a microbe or pathogen such as Escherichia coli O157:H7 (E. coli O157:H7), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterpathogenic Escherichia coli (EPEC), Salmonella, Listeria, Yersinis, Campylobacter, Clostridial species, Staphylococcus spp.; frank and opportunistic bacterial, fungal, viral, parsitic pathogens; indicator organisms including heterotrophes, generic E. coli, total and fecal conforms and enterococcus; spoilage organisms including Pseudomonas; indicator molecules including glial fibillary acid protein (GFAP), transmissable spongiform encephalopathy (TSE) agents (prions), including bovine spongiform encephalopathy (BSE) agents, scrapie, chronic wasting disease; and combinations thereof. Additional microbes or pathogens are selected from the group consisting of Staph. aureus, Bacillus cereus, and Clostridium botulinum, Clostridium perfringes, Vibrio cholerae and V. parahemolyticus, Yersinia enterocolitica, Yersinia pestis, Brucella, Francisella, Aeromonas and Plesiomonas, Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus, Providencia, Serratia, and Shigella.

[0036] Organisms that are particularly suited for testing in one or more aspects of the present invention include the following:

Bacillus anthracis
Campylobacter coli
Campylobacter jujuni
Campylobacter lari

Coliforms

[0037] E. coli 0157
E. coli, Stx-producing (STEC)
E. coli, Stx-producing with intimin
E. coli, verotoxin producing
Listeria grayi
Listeria innocua
Listeria ivanovii
Listeria monocytogenes
Listeria seeligeri

Listeria spp.

[0038] Listeria welshimeri

Salmonella spp.

[0039] Staphylococcal enterotoxins A, B, C (C1, C2, C3), D and E
Staphylococcus aureus
Yeast and mold

[0040] Each publication or patent cited herein is incorporated herein by reference in its entirety.

[0041] The foregoing description of the present invention has been presented for purposes of illustration and description. Furthermore, the description is not intended to limit the invention to the form disclosed herein. Consequently, variations and modifications commensurate with the above teachings, and the skill or knowledge of the relevant art, are within the scope of the present invention. The embodiments described hereinabove are further intended to explain the best mode known for practicing the invention and to enable others skilled in the art to utilize the invention in such, or other, embodiments and with various modifications required by the particular applications or uses of the present invention. It is intended that the appended claims be construed to include alternative embodiments to the extent permitted by the prior art.