Novel multiplex PCR assays for rapid identification of Salmonella serogroups B, C1, C2, D, E, S. enteritidis, and S. typhimurium. Issue 14 (24th March 2022) - Shang, Yuting
Abstract : Pan-genome analysis identified genes specific to Salmonella serogroups B, C1, C2, D, E, and S. enteritidis . Three multiplex PCR assays for detecting five Salmonella serogroups (B, C1, C2, D, and E) and two serovars ( S. enteritidis and S. typhimurium ) were sufficiently specific and rapid. Abstract : Foodborne illnesses caused by Salmonella represent a significant public health problem worldwide. The aim of this study was to establish multiplex PCR (mPCR) for the rapid identification of Salmonella serogroups B, C1, C2, D, and E as well as for the serovars enteritidis and typhimurium . Employing pan-genome analysis and PCR verification, B-rfbJ, C1-9679, C2-pimB, D-rfbJ, E-rfbC, and four genes ( SE18636, SE16574, SE2599, and SE13329 ) were identified as specific target genes for Salmonella serogroups B, C1, C2, D, E, and S. enteritidis, respectively. Thereafter, three novel mPCR assays (one of 3-mPCR and two of 2-mPCR) were successfully developed to identify these bacteria based on the target genes and another S. typhimurium -specific STM4495 gene. The primers targeting C1-9679, C2-pimB, and E-rfbC genes specific to the serogroups C1, C2, and E, respectively, constituted a 3-mPCR, while the other two 2-mPCRs, respectively, consisting primers specific to serogroup D and S. enteritidis ( D-rfbJ and SE16574 ), and serogroup B and S. typhimurium -specific primers ( B-rfbJ and STM4495 ), were also designed. The specificity of each mPCR was further evaluated by using non-target strains. The detection limits of mPCRs were approximately 10 3 –10 4 CFU mL −1 in pure culture and 10 4 –10 5 CFU g −1 in spiked chicken meat. In addition, mPCR assays could correctly detect target Salmonella in food samples. These results suggest that specific targets could be mined efficiently through a pan-genome analysis tool, and the novel mPCR assays developed in this study offer a promising technique for rapid and accurate detection of five serogroups of Salmonella (B, C1, C2, D, and E) and two serovars ( S. enteritidis and S. typhimurium ).