Specific Detection of Salmonella spp. in Food by Multiplex Polymerase Chain Reaction

A multiplex PCR (mPCR) assay was developed for the detection of multiple Salmonella serotypes in different kind of food products. To increase specificity of this molecular method, three sets of oligonucleotide primer were used to detect the most prevalent salmonella species. PhoP primers specific to the PhoP/PhoQ loci of coliform pathogenic bacteria such as Salmonella, Escherichia coli and citrobacter species served as presemptive indicators of enteric bacteria. Whereas Hin and Hli primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-li flagellin gene, respectively where used for specific detection of Salmonella strains. Both Hin and H-li primers are specific to motile Salmonella species and are not present in E.coli, or Citrobacter species. The traditional cultural method and serotyping method for the detection of Salmonella in food was done in parallel. The complexity of the food matrice can make Salmonella detection difficult. For this reason, phenotypic methods have been supplemented by a range of molecular-based techniques that can detect pathogen in food material in the shortest possible time. We reported here the use of a mPCR based assay for the detection of Salmonella spp. in artificially contaminated food materials. Futhermore, we have used Ampli Taq Gold polymerase (a hot start enzyme from Perkin-Elmer) that was found to be quite promessing as very consistent results were obtained using this enzyme, in almost all the food products. 74 I. Ben Salem, M. Aouni and R. Mzoughi Multiplex PCR methodology contributes to meet the need for rapid identification and detection methods in food testing laboratories. Using this assay we were able to detect all tested Salmonella serovars. The limit of detection was 1.63 10 CFU ml of pure bacterial culture. This assay could detect a different concentration of bacteria that depend on complexity of food matrices and hours of enrichment. In this study like in previous reports, Taq polymerase enzyme was routinely used and seem to succumb to the inhibitory effect of many of the food components and so the inhibition of the polymerase chain reaction . For this reason, we also tested Ampli Taq Gold polymerase (a Hot Start enzyme from Perkin-Elmer). Uses of this enzyme resolve problems of inhibitory effect of food components. The main advantage of this multiplex PCR method is its sensitivity and specificity. We also adopted a simple method DNA template isolation cutting the processing time and cost which added advantage for the use of this system in simple lab setups. In this study Ampli Taq Gold polymerase was found to be quite promising and resolve problems of inhibitory effect of food components.