Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

Abstract

In this study, we have developed a rapid method for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in foods, combining culture enrichment and a multiplex real-time polymerase chain reaction (PCR). The assay used two pre-existing primer-probe sets, labelled with different reporter dyes to enable the direct distinction of the original contaminating agent. Amplification efficiency and inclusivity/exclusivity of the combined assay was successfully assessed. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquots, and real-time PCR detection of the hly and bipA genes of L. monocytogenes and Salmonella spp., respectively. An internal amplification control, co-amplified during the PCR run, was included in the assay to verify the results. The tool was evaluated with a variety of artificially inoculated samples of fresh products and ready to eat and cooked dishes, allowing the identification of the target pathogens down to 5 CFU/25 g of food sample. Moreover, the analysis saved a considerable amount of time compared to the ISO standard, being performed in less than 2 working days. Specificity, sensitivity and accuracy were satisfactorily tested by comparison to the standard methods ISO 11290-2:1998 and ISO 6579:2002, suggesting that the tool has a great potential as a reliable alternative for food safety assurance providing rapid detection of both pathogens in food samples.

Virulence profiles of enterotoxigenic Escherichia coli isolated from piglets with post-weaning diarrhea and classification according to fecal consistency

“…Multiplex PCR improved the diagnosis, allowing the investigation of different virulence factors in the same reaction (Francis 2004), speeding results and reducing genotyping costs (Ruiz-Rueda et al 2011). Real-time polymerase chain reaction (qPCR) generate results in higher speed, sensitivity and specificity when compared to traditional PCR methods (Elizaquivel et al 2010).…”

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

“…This bacterium has the peculiarity that it can survive or even grow at low temperatures [12,13]. Ingestion of foods contaminated with L. monocytogenes can result in listeriosis, a severe infectious disease characterized by meningoencephalitis, abortion, septicemia and a high fatality rate (30%) [13][14][15][16]. The number of listeriosis cases in humans in Europe increased in 2009 by 19.1% compared to 2008, with 1,645 conWrmed cases.…”

Comparison between Listeria monocytogenes single and multipathogen detection methods

“…Most of the evaluated studies presented multipathogen detection, which means that this methodology is increasingly used and is able to detect many foodborne pathogens at the same time. This is probably consequence of some of the advantages of this type of analyses, namely the consumption of fewer raw materials and the ability of cover a broad spectrum of potential pathogens [14,15] . This is important because it may permit the detection of a higher number of microorganisms simultaneously, foreseeing different foodborne diseases and ensuring product quality and safety.…”

“…A maioria dos estudos avaliados apresentou detecção de multipatógenos, o que significa que esta metodologia é cada vez mais utilizada e é capaz de detectar muitos patógenos transmitidos pelo consumo de alimentos contaminados. Esta é provavelmente uma consequência de algumas das vantagens deste tipo de análise, nomeadamente o consumo de menos matéria-prima e a capacidade de cobrir um espectro mais amplo de potenciais agentes patogênicos [14,15] . Isto é importante porque pode garantir a detecção de um número mais elevado de microrganismos simultaneamente, prevendo diferentes doenças transmitidas por alimentos e garantindo a qualidade e segurança dos produtos.…”