In-house validation of a real-time PCR method for rapid detection of Salmonella ssp. in food products

Abstract

A real-time polymerase chain reaction (PCR) method for Salmonella ssp. detection in food samples has been developed and validated in-house. The specificity of the assay was confirmed by tests with 295 different Salmonella strains, including four strains of Salmonella bongori. When tested with extracted Salmonella DNA the lowest detected amount was found to be 5 fg, which is equivalent to approximately one genome copy. The detection limit was further determined by artificial contamination of minced meat with S. Typhimurium cells and of pastry with S. enteritidis using the most probable number approach for cell dose dilutions. It was calculated that even one colony forming unit of Salmonella was still detectable in 25 g food after enrichment culture for 18 h. An additional PCR system for internal positive control, which was included in each reaction and detected in parallel via another reporter fluorescence dye, has no negative impact on the sensitivity of the assay. The method was evaluated with 1,293 naturally contaminated food samples and compared to the conventional cultural method. Of 55 positive PCR samples, 45 were confirmed by the cultural method. The statistical comparison revealed a correlation of 99.2% for specificity, of 100% for sensitivity and of 99.2% for trueness. The results of the comparative analysis and the advantages of the real-time PCR method for detection of Salmonella ssp. under routine laboratory conditions are discussed.