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 AmpliWcation 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 modiWed by adding Tween 80 was used for the enrichment step. SpeciWcity of the method was checked against a panel of 24 non-target bacterial strains. RT-PCR eYciency obtained for the simultaneous ampliWcation 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 diVerent 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 Wrst 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 diVerent processed food samples with a low LOD. The application of this method can signiWcantly reduce costs and time of analysis in laboratories, what would be reXected in a faster response in those risk situations when they are detected.

Detection of foodborne pathogens by qPCR: A practical approach for food industry applications

“…Obtained results were similar to traditional culture methods or even lower, with values in general below 10 CFU/25 g, with the added advantage that, with multiplex qPCR, several targets were detected simultaneously Garrido, Chapela, Román, et al, 2012;Garrido, Chapela, Román, Fajardo, Lago, et al, 2013). Low levels of target pathogens can also be detected in food matrices contaminated by other dominant micro-organisms.…”

“…When quantifying pathogens in food, the lower LOQ in the food matrix should be considered and not the lower LOQ obtained from pure cultures because it takes into account the efficiency of nucleic acid extraction and possible interactions of food components with PCR amplification. In agreement with EN ISO 22174:2005, a standard for PCR application in order to detect foodborne pathogens, and with experts in PCR/qPCR ISO, 2005), an increasing number of studies have included an internal amplification control to qPCR protocols, to avoid negative deviations (Calvó, Martínez-Planells, Pardos-Bosch, & Garcia-Gil, 2008;Garrido, Chapela, Román, et al, 2012;Garrido, Chapela, Román, Fajardo, Lago, et al, 2013;Jofré et al, 2005;Nordstrom, Vickery, Blackstone, Murray, & DePaola, 2007).…”

“…In contrast, when a positive result is found, the duration of the analyses is greatly extended as additional isolation and identification steps must be performed. However, recent studies have demonstrated that times can be significantly shortened to only 24 h by optimizing qPCR methods for most of these bacteria (Chapela et al, 2010;Garrido, Chapela, Román, et al, 2012;Garrido, Chapela, Román, Fajardo, Lago, et al, 2013).…”

Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay

“…Most PCR assays for the detection of Shigella in food are based on the sole detection of ipaH and cannot differentiate between natural isolates or cross-contamination with the genetically engineered S. flexneri 2457M FDA positive control strain (Mokhtari et al, 2013;Garrido et al, 2013;Lin et al, 2010;Wang et al, 2007;Warren et al, 2006). Accidental in-laboratory sample contamination with 2457M could generate a false-positive result that could lead to regulatory action and/or the destruction of the tested food.…”

Multiplex real-time PCRs for detection of Salmonella, Listeria monocytogenes, and verotoxigenic Escherichia coli in carcasses of slaughtered animals

“…The ISO standards for Salmonella, L. monocytogenes, and VTEC O157 detection involve sample culture in pre-enrichment and selective media, followed by confirmation of bacterial isolates according to their morphological, biochemical, and immunological characteristics (10)(11)(12)(13). These methods, although reliable and relatively inexpensive, are laborious and require several days before the analysis is completed (5). Furthermore, the obtained results often are not available before the food has been either released for trading or consumed, which may result in the spread of pathogens (15).…”