Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidium monoazide and real-time PCR

The increase of foodborne outbreaks associated with fresh vegetables has highlighted the importance of developing rapid and specific methods for the detection and quantification of foodborne pathogens. In this sense, real-time PCR (qPCR) fulfills these requirements although it may detect dead cells. Recently, a potential strategy to specifically detect viable cells has been proposed relying on the use of DNA binding molecules as sample pretreatment previous to the qPCR. In this study propidium monoazide (PMA) and reagent D, combined with qPCR, were evaluated for the detection and quantification of viable Escherichia coli O157:H7, Salmonella and Listeria monocytogenes. Initially, the optimal concentration of both reagents was determined for discrimination between viable and dead bacteria in cell suspensions. Although both reagents showed similar reductions for the three pathogens, reagent D was toxic to L. monocytogenes and Salmonella and therefore only PMA was used to evaluate the applicability of this technique on food samples. A final concentration of 50 μM PMA was assayed in artificially inoculated spinach and mixed salad. PMA-qPCR signal was negative for all dead cell concentrations tested except for mixed salad inoculated with L. monocytogenes at the highest concentration. These results demonstrate that PMA-qPCR is a suitable technique for the detection and quantification of viable pathogens in fresh-cut vegetables at the levels normally found in vegetable samples.