Detection of Salmonella enterica in food using two-step enrichment and real-time polymerase chain reaction

Aims: A new real-time polymerase chain reaction-based method was developed for the detection of Salmonella enterica in food. Methods and Results: The method consisted of a novel two-step enrichment involving overnight incubation in buffered peptone water and a 5-h subculture in Rappaport-Vassiliadis medium, lysis of bacterial cells and a Salmonella-specific 5'-nuclease real-time PCR with an exogenous internal amplification control. Because a two-step enrichment was used, the detection limit for dead S. enterica cells in artificially contaminated ice cream and salami samples was high at 10(7) CFU (25 g)(-1), eliminating potential false-positive results. When the method was evaluated with a range of 100 naturally contaminated food samples, three positive samples were detected by both the real-time PCR-based method and by the standard microbiological method, according to EN ISO 6579. When the real-time PCR-based method was evaluated alongside the standard microbiological method according to EN ISO 6579 with 36 food samples artificially contaminated at a level of 10(0) CFU (25 g)(-1), identical results were obtained from both methods. Conclusions: The real-time PCR-based method involving a two-step enrichment produced equivalent results to EN ISO 6579 on the day after sample receipt. Significance and Impact of the Study: The developed method is suitable for rapid detection of S. enterica in food.