Multiplex high resolution melt-curve real-time PCR assay for reliable detection of Salmonella

A highly specific multiplex high resolution melt-curve (HRM) real-time PCR assay was developed for the reliable detection of Salmonella serotypes in general. As compared to a conventional singleplex PCR, the use of HRM and three specific gene targets allowed for the detection of the majority of Salmonella serotypes while reducing the chances of generating false-negative or false-positive results from non-Salmonella strains. Primers for each of the three target genes, invA, stn and fimA, were specifically designed. This method was validated with 81 Salmonella strains and its applicability was tested on a wide range of food samples. HRM-PCR results showed three specific and well separated peaks for most Salmonella serotypes, which indicated specific amplification of all three target genes. Several Salmonella strains showed the absence of one peak, but gave positive results for the other two target genes. Twenty-nine non-Salmonella isolates yielded negative results for all three genes, while Citrobacter freundii was positive for the stn gene. After a 6-h enrichment, target genes could be detected in 25 g of most of the 24 different food samples inoculated with 103 CFU/g of Salmonella.