Multiplexed Stn and Plca based specific genetic marker for early detection of Salmonella enterica and Listeria monocytogenes in milk samples

Abstract

In this study conventional PCR and multiplex PCR based method was developed for the detection of two common foodborne pathogens Salmonella enterica and Listeria monocytogenes in 20 raw milk samples. The PCR was undertaken to detect two genes namely, Salmonella enterotoxin (stn) gene and phosphatidylinositol-specific phospholipase C gene (plcA) from both the organisms. The DNA templates (for both organisms) were amplified using specific set of primers. The resulting amplicons were found to be 265 bp and 147 bp respectively. Validation studies were further performed in artificial spiked milk samples and raw milk samples using multiplex PCR. The available detection methods are bacterial culturing, biochemical tests, serological tests, antibiotic sensitivity, ELISA and PCR. All these methods are either expensive or non-confirmatory and have some limitations. The reported multiplexed PCR based genetic marker completes overall analysis in 80 min which is the minimum time reported so far for the confirmation of these foodborne pathogens. Sensitivity and specificity of developed method was calculated and compared with different conventional methods. The detection limit of the assay for the S. enterica was 6.6 x100 CFU/mL and for L. monocytogenes was 4.5x100 CFU/mL.