Comparative performance of two different detection chemistries of qPCR for their implementation in a same-day detection method to determine the presence of Shiga toxin-producing Escherichia coli in ready-to-eat salads

Shiga toxin-producing E. coli (STEC), are among the most frequently reported foodborne pathogens worldwide. They produce two powerful cytotoxins, called Shiga toxins (Stx1 and Stx2). Most DNA-based methods target these genes, and rely on lengthy enrichment protocols which delay early identification of potential threats. Vegetable samples are particularly problematic, as they are rich in components that may inhibit these techniques. In the present study, a short enrichment and improved matrix lysis protocols were combined and tested with qPCR implementing two different detection chemistries, a hydrolysis probe, and an intercalating dye-based one taking advantage of SYBR Green, to determine which performed best. The methodology presented a turnaround time of -6 h including the enrichment, sample treatment followed by DNA extraction, and STEC detection with either detection chemistry. Minor differences were identified among both detection chemistries, as the LOD95 were 6.9 and 8.6 CFU/25 g for the probe-based and intercalating dye respectively; both got relative sensitivities, specificities, and accuracies of 100 % and a Cohen's kappa, of 1.00, being this an "almost complete concordance" with the expected result. Overall, same-day detection of STEC in ready-to-eat salad samples, was achieved, and both qPCR detection chemistries demonstrated suitable for the intended application.