Detection of Shiga toxin genes stx1, stx2, and the +93 uidA mutation of E. coli O157:H7/H-using SYBR® Green I in a real-time multiplex PCR

Enterohemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen capable of causing diarrhea and vomiting, but more serious complications such as hemorrhagic colitis and hemolytic-uremic syndrome (HUS) can result. A real-time PCR method to detect the presence of Shiga toxin producing E. coli (STEC) and E. coli O157:H7 was investigated using SYBR® Green I (SG). Primers were designed to target the Shiga toxin genes (stx1 and stx2) and a highly conserved base substitution at +93 of the β-glucuronidase gene (uidA) unique to E. coli O157:H7. An initial test panel of five E. coli and non-E. coli isolates was tested with individual primer sets (simplex assay) and all primer sets including stx1, stx2, and uidA(multiplex assay). All strains were correctly identified in both assays. Average melt temperatures (Tm's, °C) for PCR products were 85.42-stx1, 81.93-stx2, and 88.25-uidA in simplex assays and 85.20-stx1, 81.20-stx2, and 88.16-uidA when multiplexed. Each of the three gene targets in one multiplex reaction could be distinguished by melt curve data with significantly different Tm's. The assay was expanded to a panel of 138 isolates consisting of STEC, E. coli O157:H7, non-toxigenic E. coli, and non-E. coli isolates with melt peaks consistent with those stated above.