A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory

ABSTRACTA multiplex PCR was developed for the detection of the following genes characteristic of diarrhoeagenic Escherichia coli (DEC): verocytotoxins 1 (vtx1) and 2 (vtx2), characteristic of verocytotoxin-producing E. coli (VTEC); intimin (eae), found in enteropathogenic E. coli (EPEC), attaching and effacing E. coli and VTEC; heat-stable enterotoxin (estA) and heat-labile enterotoxin (eltA), characteristic of enterotoxigenic E. coli (ETEC); and invasive plasmid antigen (ipaH), characteristic of enteroinvasive E. coli (EIEC) and Shigella spp. The method allowed the simultaneous identification of all six genes in one reaction, and included a 16S rDNA internal PCR control. When applied to pure cultures from a reference strain collection, all virulence genes in 124 different DEC strains and 15 Shigella spp. were identified correctly, and there were no cross-reactions with 13 non-E. coli species. The detection limit of the method was 102–103 DEC CFU/PCR in the presence of 106 non-target cells. When the multiplex PCR was tested with colonies from plate cultures of clinical stool samples, it was a faster, more sensitive, less expensive and less laborious diagnostic procedure than DNA hybridisation. When used with DNA purified from spiked stool samples (by two different commercial kits), the method had a detection limit of 106 CFU/mL stool sample.