DNA Extraction and Detection of Virulence Genes in *E. coli* Isolates

One hundred (100) presumptive *E. coli* isolates were randomly selected and inoculated separately into 5 mL Erlenmeyer flasks containing 2 mL nutrient broth (Merck, Johannesburg, South Africa). The flasks were incubated overnight at 37 ◦C on a rotary shaker at 100 rpm. DNA was extracted from 1 mL of the overnight culture using the InstaGeneTM Matrix (Bio-Rad Laboratories, Johannesburg, South Africa) following the manufacturer's instruction. The template DNA was stored at −20 ◦C for PCR assays. All selected samples were first confirmed as *E. coli* by testing for the presence of the malate dehydrogenase (*mdh*) gene which is found in most *E. coli* strains [21]. After that, the presence of a total of eight VGs (*eaeA* (EPEC/EHEC), *eagg* (EAEC), *ipaH* (EIEC), *ST* (ETEC), *ibeA* (NMEC), *stx1* (EHEC), *stx2* (EHEC) and *flicH7* (EHEC)) were investigated. The primer sequences and the PCR-cycling conditions for the identification of the various VGs were as previously described by Abia et al. [19]. Both multiplex and singleplex PCR assays were performed for the target genes. Multiplex PCR assays were divided into 3 sets where set 1 contained *eaeA*, *eagg* and *ipaH*, set 2 contained *flicH7* and *Stx1* and finally set 3 contained *ST* and *ibeA* genes [19,22,23]. Singleplex real-time PCR assays were performed for the *mdh* and *stx2* target genes [24,25].
