Simultaneous detection of Listeria monocytogenes and Salmonella spp. in dairy products using real time PCR-melt curve analysis.

Abstract

The present investigation reports development of post real time PCR (RTi-PCR) - melt curve analysis for simultaneous detection of Listeria monocytogenes and Salmonella spp. The optimal Sybr Green I (SG-I) concentration of 1.6 μM resulted in two specific peaks with melting temperature (Tm) of 79.90 ± 0.39 °C and 86.29 ± 0.13 °C for L. monocytogenes and Salmonella spp respectively. The detection sensitivity of the assay in reconstituted non-fat dried milk (NFDM; 11%) spiked with the target pathogens at different levels was 3 log cfu per ml of each pathogen. However, the sensitivity was improved up to 1 log cfu per ml by including pre-enrichment step of 6 h. On application of assay on 60 market samples, one sample each of raw milk and ice cream was detected positive for L. monocytogenes and Salmonella spp. Assay was quite specific as no cross reactivity with non target cultures could be observed. The developed assay can find valuable application in monitoring dairy products for the presence of L. monocytogenes and Salmonella spp. to ensure their microbiological quality and safety.