Detection of extended-spectrum beta-lactamase cefotaxime resistance and virulence genes in Escherichia coli by duplex quantitative real-time PCR and melt curve analysis.

Emerging virulent and antibiotic-resistant pathogens present a global public health risk. Routine monitoring of prevalence within the clinical, environmental and food production setting is vital. Quantitative real-time PCR (qPCR) coupled with melting curve analysis can rapidly and accurately characterize pathogens. We evaluated commercial qPCR mixes based on SYBR Green l and EvaGreen for developing an assay for simultaneously detecting antibiotic resistance (extended-spectrum beta-lactamase, ESBL and blaCTX-M ) and virulence (stx1, stx2 and eae) genes in Escherichia coli (n = 12) isolated from irrigation water and irrigated vegetables. SYBR Green and EvaGreen detected two amplicons (stx1 and blaCTX-M ) and (stx2 and eae) in a single reaction. A higher mean melting temperature (Tm ) separation between targeted amplicons and smoother melting curves were observed with the EvaGreen suggesting better performance when targeting multiple amplicons. Through simple stepwise optimization of DNA, cycling, primers, reaction volume and melting curve scanning rate, we adopted a conventional PCR assay for detection of large amplicons (375-1580 bp) for qPCR. This may facilitate development of cost-effective tailor-made assays for rapid and accurate monitoring of emerging foodborne and environmental pathogens in resource constrained regions.