Multiplex Real-time PCR Assay for Reliable Detection of Salmonella

Introduction: Due to a large number of serotypes and frequent mutations of Salmonella virulence genes, finding a mutual gene to detect all Salmonella serotypes in one assay is impossible. In addition, some non-Salmonella strains, such as Citrobacter amalonaticus, will yield false-positive results with single gene detections.

Purpose: The aim of this study was to design a three-gene multiplex high resolution melt-curve (HRM) PCR to detect the majority of Salmonella serotypes while avoiding false-positive and false-negative results.

Methods: The invA, stn and fimA genes were selected due to their widespread presence in all Salmonella strains. Primers were designed and tested for each target gene. A melt curve with an internal amplification control was standardized in the assay. This method was tested on 40 Salmonella strains and applied in various food samples.

Results: Three specific and separated peaks were formed for most Salmonella serotypes in this PCR, which indicated that all three products were formed. Several Salmonella strains showed the absence of one peak, but gave positive results for the other two target genes. After a 6-h enrichment, target genes could be detected in 25 g of food samples inoculated with 10³ CFU of Salmonella.

Significance: This assay uses a HRM multiplex PCR rather than a singleplex PCR to detect Salmonella serotypes, which reduces the chance of getting false-negative results from Salmonella serotypes missing one of the target genes, and avoids false-positive results from non-Salmonella strains that possess one of the three genes, making it a highly reliable and accurate method to detect Salmonella in foods.