Semi-automated fluorogenic PCR assays (TaqMan) for vapid detection of Escherichia coli O157 : H7 and other Shiga toxigenic E. coli

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 designe d to amplify 80, 120 and 150 bp regions of virulence genes stx1, stx2 and e aeA, 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 thre e sets of primers and fluorogenic probes were included in one reaction to s imultaneously amplify and detect any of the three targeted virulence genes. In non-multiplex PCR assay, each of the three virulence genes was amplifie d and detected in independent reactions. The specificity of these assays wa s evaluated using suspensions of STEC and other bacterial species lacking s tx1, stx2 and eaeA. The multiplex assay detected all STEC harbouring any co mbination of th ree virulence genes. Three non-multiplex PCR reactions iden tified 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 wh en performed simultaneously or within 13 h if the multiplex assay is used a s 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.