title: Detection of enterohemorrhagic Escherichia coli O157:H7 by using a multiplex real-time PCR assay for genes encoding intimin and Shiga toxins.
abstract: A multiplex real-time PCR (R-PCR) assay was designed and evaluated on the ABI 7700 sequence detection system (TaqMan) to detect enterohemorrhagic Escherichia coli (EHEC) O157:H7 in pure cultures, feces, and tissues. Three sets of primers and fluorogenic probes were used for amplification and real-time detection of a 106-bp region of the eae gene encoding EHEC O157:H7-specific intimin, and 150-bp and 200-bp segments of genes stx1 and stx2 encoding Shiga toxins 1 and 2, respectively. Analysis of 67 bacterial strains demonstrated that the R-PCR assay successfully distinguished EHEC O157:H7 serotype from non-O157 serotypes and provided accurate profiling of genes encoding intimin and Shiga toxins. Bacterial strains lacking these genes were not detected with this assay. The detection range of the R-PCR assay for the three genes was linear over DNA concentrations corresponding from 10(3) to 10(8)CFU/ml of EHEC O157:H7. The R-PCR allowed construction of standard curves that facilitated quantification of EHEC O157:H7 in feces and intestinal tissues. Detection sensitivity of the R-PCR assay ranged from 10(4) to 10(8)CFU/g of feces or tissues without enrichment. Enrichment of feces in a non-selective broth for 4 and 16h resulted in the detection of levels (from 10(0) to 10(3)CFU/g of feces) considered sufficient for infection in humans. The R-PCR assay for eae(O157:H7), stx1, and stx2 proved to be a rapid test for detection of EHEC O157:H7 in complex biological matrices and could also potentially be used for quantification of EHEC O157:H7 in foods or fecal samples.
authors.affiliation: Pre-Harvest Food Safety and Enteric Diseases Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA 50010, USA. vsharma@nadc.ars.usda.gov
authors.forename: Vijay K
authors.initials: VK
authors.lastname: Sharma
authors.forename: Evelyn A
authors.initials: EA
authors.lastname: Dean-Nystrom
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology*; D002432:Cecum / Q000382:microbiology; D015169:Colony Count, Microbial; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004927:Escherichia coli Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D019453:Escherichia coli O157 / Q000235:genetics* / Q000302:isolation & purification; D029968:Escherichia coli Proteins; D005243:Feces / Q000382:microbiology; D007082:Ileum / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1 / Q000235:genetics* / Q000378:metabolism; D022641:Shiga Toxin 2 / Q000235:genetics* / Q000378:metabolism; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000382:microbiology*
chemical_list: D018829:Adhesins, Bacterial; D002352:Carrier Proteins; D015342:DNA Probes; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; C073819:eaeA protein, E coli