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...

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