A new multiplex real-time PCR developed method for Salmonella spp. and Listeria monocytogenes detection in food and environmental samples

Despite efforts done by industries some well known foodborne pathogens, like Salmonella spp. and Listeria monocytogenes, continue to be a challenge to public health institutions and a threat for consumers. The aim of this study was to develop a complete, rapid and reliable multiplex real-time PCR (qPCR) method for the simultaneous detection of these two bacteria in food and environmental samples, including a novel single enrichment broth (TA10) for both bacteria. TA10 broth was modified (pH and buffer concentration) to enhance simultaneous growth of both pathogens in the presence of high numbers of competitors bacteria. Also two different DNA-extraction protocols were compared. qPCR efficiency above 90% was obtained, covering 5 orders of magnitude. Complete method achieved low limit of detection (5 cfu/25 g), and all quality parameters of the method returned values over 90%. Complete qPCR method was applied to 95 natural samples covering a wide variety of food types proving that the qPCR method described, including the use of one single enrichment broth, modified TA10, was suitable for the simultaneous and reliable screening of Salmonella spp. and L. monocytogenes in food and environmental samples.