title: Semi-automated fluorogenic PCR assays (TaqMan) forrapid detection of Escherichia coli O157:H7 and other shiga toxigenic E. coli.
abstract: Semi-automated detection of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 and non-O157:H7 Shiga toxin-producing E. coli (STEC) was achieved using fluorogenic polymerase chain reaction (PCR). These PCR assays were designed to amplify 80, 120 and 150 bp regions of virulence genes stx1, stx2 and eaeA, respectively, using specific primers. The fluorogenic probes were used for specific detection of amplified products of the stx1 and stx2 genes of STEC, and the eaeA gene of EHEC O157:H7. For multiplex PCR assay, the three sets of primers and fluorogenic probes were included in one reaction to simultaneously amplify and detect any of the three targeted virulence genes. In non-multiplex PCR assay, each of the three virulence genes was amplified and detected in independent reactions. The specificity of these assays was evaluated using suspensions of STEC and other bacterial species lacking stx1, stx2 and eaeA. The multiplex assay detected all STEC harbouring any combination of three virulence genes. Three non-multiplex PCR reactions identified types of Shiga toxin genes carried by a STEC and identified STEC as either EHEC O157:H7 or non-O157:H7 STEC. Sensitivity limits of these assays in beef and faeces inoculated with EHEC O157:H7 were 5.8 to 580 cfu and 1.2 to 1200 cfu, respectively. These assays can be completed within 8-10 h when performed simultaneously or within 13 h if the multiplex assay is used as an initial screen for detecting STEC and the non-multiplex assay is used for subsequent detection of stx1 and stx2 of STEC and eaeA of EHEC O157:H7
authors.affiliation: Enteric Diseases and Food Safety Research Unit, National Animal Disease Center, USDA, Ames, Iowa, 50010, USA. vsharma@nadc.ars.usda.gov
authors.forename: V K
authors.initials: VK
authors.lastname: Sharma
authors.forename: E A
authors.initials: EA
authors.lastname: Dean-Nystrom
authors.forename: T A
authors.initials: TA
authors.lastname: Casey
mesh_terms: D018829:Adhesins, Bacterial; D000818:Animals; D001331:Automation; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D002352:Carrier Proteins; D002417:Cattle; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D029968:Escherichia coli Proteins; D005243:Feces / Q000382:microbiology; D005453:Fluorescence; D006801:Humans; D008460:Meat / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D022603:Shiga Toxins; D013997:Time Factors
chemical_list: D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001427:Bacterial Toxins; D002352:Carrier Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D022603:Shiga Toxins; C073819:eaeA protein, E coli