Detection of virulence and extended spectrum β-lactamase genes in Salmonella by multiplex high-resolution melt curve real-time PCR assay.

AIMS Develop and standardize multiplex high-resolution melt curve (HRM) real-time PCR assays for simultaneous detection of Salmonella virulence and extended spectrum β-lactamase (ESBL) genes in food. METHODS AND RESULTS Two sets of multiplex real-time PCR assays targeting six virulence and three ESBL genes with internal amplification control were standardized. The first assay detected hilA, fimH, sipA, blaTEM and blaSHV, and the second detected invA, fimA, stn and blaCMY . The PCR assays were validated with DNA samples from 77 different Salmonella strains. The assay specificity was tested with DNA from 47 non-Salmonella strains. Melt curve analyses showed distinct, well-separated melting peaks of each target gene detected by their respective melting temperatures (Tm ). Different food samples were spiked with 10, 102 and 103  CFU/ml of Salmonella. The optimized assays were able to detect all target genes in concentrations of as low as 10 CFU/ml in 25 g foods within 10 h of enrichment. CONCLUSIONS Multiplex HRM real-time PCR assays can be used as rapid detection methods for detecting Salmonella in foods. SIGNIFICANCE AND IMPACT OF STUDY The assays developed in this study will allow for accurate detection of virulence and ESBL genes in Salmonella that are present in low concentrations in food samples.