Molecular beacon based real-time PCR assay for simultaneous detection of Listeria monocytogenes and Salmonella spp. in dairy products

In this study, a molecular beacon-based duplex real-time PCR assay was developed for the simultaneous detection of Listeria monocytogenes and Salmonella spp. by targeting their virulence genes hly and invA, respectively. The detection sensitivity of the assay in reconstituted non-fat dried milk (11%) was 3 and 4 log cfu mL(-1) for L. monocytogenes and Salmonella spp., respectively, without any pre-enrichment. However, after pre-enrichment of the samples in brain heart infusion broth for 6 h, the assay could detect as low as 1 log cfu of both the pathogens. The assay was quantifiable for the respective pathogens over 5 and 4 log cfu mL(-1) with regression coefficient of 0.9956 and 0.9905. On application of the developed assay on 60 dairy products, one sample of raw milk was positive for L. monocytogenes, while Salmonella spp. was detected in one sample of ice cream. The performance of the duplex assay was validated using microbiological methods and commercial individual pathogen detection kits, which further affirmed the performance of the assay by positively detecting the same samples. The developed assay can be used to monitor the quality of dairy products in context of L. monocytogenes and Salmonella spp.