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.

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.