In this study, duplex real-time PCR approach with melting curve analysis is presented for the detection of Listeria monocytogenes, which are important food-borne bacterial pathogens usually present in imported frozen meat. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic Tm values demonstrating the specific and efficient amplification of the two pathogens;73.2°C and 81.54°C for L. monocytogenes and Salmonella spp., respectively, the duplex, real-time PCR was equally sensitive to uniplex real-time PCR, using same amounts of purified DNA. When our established duplex real-time PCR assay was applied to artificially inoculated imported frozen meat the detection limit was 103 CFU/g for each of these pathogens without enrichment. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquot. The results from this study showed that the developed duplex real-time PCR with melting curve analysis is promising as a rapid and cost-effective test method for improving food safety.
Key words: Salmonella, Listeria monocytogenes, Multiplex, Real-time PCR
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