A single step multiplex PCR for identification of six diarrheagenic E. coli pathotypes and Salmonella.

Abstract

E. coli is generally a commensal but includes some highly pathogenic strains carrying additional genes in plasmids and/or the chromosome. Based on these genes the pathogenic strains are divided into pathotypes including enteropathogenic (EPEC), enterohemorrhagic (EHEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), enteroinvasive (EIEC) and diffusely adherent (DAEC) E. coli. Here, previously developed multiplex PCR strategies for these strains were integrated into one single step multiplex that differentiates all these E. coli pathotypes, usually based on multiple characteristic PCR products. This multiplex PCR works reliably for colony PCR. Two additional markers were added: one to detect most Enterobacteriacea, which acts as a positive control for successful PCR, and one to distinguish Salmonella. The multiplex correctly classified a set of 45 reference strains by colony PCR and 71 (45+26) strains by in silico PCR. It was then used to interrogate 44 clinical strains from bovine hosts resulting in detection of EAEC and DAEC determinants.