Development and Evaluation of a Multiplex Fluorescence PCR for Salmonella Virulence Genes Analysis

Background: Salmonellosis, a disease caused by Salmonella, is a significant public health concern and economic burden worldwide. The ability of various Salmonella serovars to cause disease is closely linked to the virulence genes they possess. Objectives: The aim of this study was to develop a multiplex fluorescence PCR for detecting ten major virulence genes (ssaR, spvC, pefA, sipA, fimA, sifA, sopE2, sopB, prgH, and stn) in Salmonella. Methods: Primer pairs specific to ten target virulence genes were designed using Primer Premier 5.0 and distributed across two reaction tubes. The multiplex fluorescence PCR was optimized by adjusting one factor at a time. Results: A total of sixty Salmonella strains were analyzed using the newly developed multiplex fluorescence PCR. All strains contained seven or more of the tested virulence genes. The positive rates of virulence genes ssaR, sipA, sopE2, sopB, prgH, and stn were high, each at 100%. The positive rate of sifA was also relatively high at 81.67%. However, the positive rates of spvC at 5% and pefA at 3.33% were relatively low. Conclusions: The newly developed multiplex fluorescence PCR provides a straightforward, cost-effective, and high-throughput solution for detecting virulence genes in Salmonella. It has the potential to become a routine method for analyzing Salmonella virulence genes.