The second assay detects Shiga-toxin (stx) genes, which are associated with virulence in Escherichia coli and Shigella spp., and differentiates the subtypes of stx-1 and stx-2 based on SNP loci.
Whereas isolates of the "Top-6" serotypes (O26, O45, O103, O111, O121, O145) had a high incidence of stx1a-I (90%) and stx2a (100%). stx1a-III (60%) was only observed in non Top-7 (Top-6 plus O157) STEC and Shigella spp.
The virulence genes selected for each category were Stx1, Stx2, and eaeA for enterohemorrhagic E. coli (EHEC), eaeA for enteropathogenic E. coli (EPEC), STIb and LTI for enterotoxigenic E. coli (ETEC), ipaH for enteroinvasive E. coli (EIEC) and Shigella spp., and aggR for enteroaggregative E. coli (EAEC).
The targets selected for each group were eae and bfpA for EPEC isolates, the target of probe CVD432 for EAEC isolates, the genes encoding heat-labile and heat-stable toxins for ETEC isolates, stx(1) and stx(2) for STEC isolates, and ipaH for EIEC isolates and Shigella spp.