Development of a multiplex real-time PCR method for simultaneous detection of Salmonella enterica, Shigella flexneri and Listeria monocytogenes in processed food samples

Abstract

The present work is focused on the development of a TaqMan multiplex real-time PCR method for the detection of Salmonella, Shigella and L. monocytogenes in seafood, meat and ready-to-eat products. The aim of this study is to detect the three pathogens in one single test including an enrichment medium for the simultaneous growth of the bacteria of interest and an Internal Amplification Control (IAC) to monitor PCR inhibitors. For this purpose, three genes were selected, invA for Salmonella, ipaH for Shigella and hlyA for L. monocytogenes. Also, no. 17 broth without dextrose and further modified by adding Tween 80 was used for the enrichment step. Specificity of the method was checked against a panel of 24 non-target bacterial strains. RT-PCR efficiency obtained for the simultaneous amplification of all three pathogens was 102.5% for Salmonella, 108.9% for Shigella and 106.4% for L. monocytogenes. The limit of detection (LOD) was evaluated in seafood, meat and ready-to-eat products, being established within 3 and 22 cfu in 25 g of sample for the three bacteria analyzed. Seventy-eight samples were analyzed with multiplex RT-PCR including spiked and natural samples collected from different laboratories. Even though several RT-PCR methods have been developed for the detection of Salmonella, Shigella and L. monocytogenes, as far as we know this is the first method developed for the simultaneous detection of these three pathogens, coupling RT-PCR with an enrichment in the same broth and being tested in a wide range of different processed food samples with a low LOD. The application of this method can significantly reduce costs and time of analysis in laboratories, what would be reflected in a faster response in those risk situations when they are detected.