Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples

Abstract

The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for specificity, and 124 Salmonella spp. including type strains and 116 non- Salmonella strains were evaluated. All Salmonella strains tested were invA-positive and all non- salmonella strains yielded no amplification products. The melting temperature ( T m =79 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the threshold cycle ( C T) versus copy numbers of Salmonella Enteritidis showed good linearity in broth ( R 2=0.994; slope=3.256) and sterilized milk ( R 2=0.988; slope=3.247), and the minimum levels of detection were >10 2 and >10 3 colony forming units (CFU)/ml, respectively. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated samples. Lagoon water, feed/silage, bedding soil, and bulk tank milk samples obtained from dairy farms were spiked with 10 0 to 10 5 CFU/ml of Salmonella Enteritidis. Sensitivities for detecting Salmonella in these sources were 10 3 to 10 4 CFU/ml of inoculums in broth without enrichment. Detection limits were reduced to <10 CFU/ml of inoculum in broth after 18 h enrichment. Ninety-three environmental samples including fecal slurry, feed/silage, lagoon water, drinking water, bulk tank milk, farm soil, and bedding soil were analyzed for the presence of Salmonella by real-time PCR, results were compared with those obtained by conventional culture methods. All samples analyzed were negative for Salmonella by both real-time PCR and standard culture method. No false positive or false negative results were detected.