Multiplex PCR Assay for Virulence Genes

The presence of eight specific VAGs (papC, iucD, irp2, tsh, vat, astA, iss, and cva/cvi) was tested by a multiplex PCR [9 (link)]. The virulence-associated genes encode P-fimbria associated with adhesion [16 (link)], iron acquisition systems (iucD and irp2) [24 (link),25 (link)], and the temperature-sensitive autotransporter protein associated with high virulence [26 (link)]. The vat gene encodes a cytotoxic vacuolating autotransporter protein [27 (link)] the enteroaggregative heat-stable enterotoxin (astA) [28 (link)] a protein for increased serum survival (iss) [29 (link)] and the plasmid-borne genes cva/cvi which cause disruption of the membrane of sensitive cells [30 (link)]. E. coli isolated from MacConkey without any antimicrobials was used (Oxoid, CM0007). In brief, DNA was extracted by using a Maxwell^® RSC Instrument and Cultured Cells DNA kits (AS1620, Maxwell, Nacka, Sweden), as recommended by the manufacturer. PCR running conditions [9 (link)] were followed, except that agarose gels were run for 80 min at 75 V. A GeneRuler 100 bp plus DNA ladder (Thermo Scientific, SM0323, Vilnius, Lithuania) was used as a size marker on each gel.