Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli

A multiplex PCR-DNA probing assay was developed to detect four major Escherichia coli virotypes. Six highly specific polymerase chain reaction (PCR) primer sets and DIG-labeled chemiluminescent probes were designed to target the Shiga-like toxin I and II genes (stxI and stxII) of verotoxigenic E. coli (VTEC), heat-stable and heat-labile toxin genes of enterotoxigenic E. coli (ETEC), adherence factor (EAF) of enteropathogenic E. coli (EPEC) and a fragment of the invasiveness plasmid (IAL) of enteroinvasive E. coli (EIEC). The primer pairs generate products of 350, 262, 170, 322, 293 and 390 bp in length, respectively. The multiplex primers and probes were tested for specificity against 31 pathogenic E. coli strains, nine nonpathogenic E. coli and non-E.coli enteric and environmental bacterial strains. The results showed a high degree of specificity of the primers and probes for strains from corresponding virotypes and no reaction with the nontarget bacterial strains. The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major virotypes of E. coli.