Development and evaluation of a dual priming oligonucleotide system-based multiplex PCR assay for simultaneous detection of six foodborne pathogens

In this study, a multiplex PCR assay with dual priming oligonucleotide system (DPO system-based mPCR) was developed for the simultaneous detection of Salmonella spp., Listeria (L.) monocytogenes, Shigella spp., Staphylococcus (S.) aureus, Campylobacter (C.) jejuni and Yersinia (Y.) enterocolitica from food. Pathogen-specific DPO systems were designed targeting Salmonella spp. fimY gene, L. monocytogenes iap gene, Shigella spp. ipaH gene, S. aureus 442 gene, C. jejuni gyrA gene and Y. enterocolitica 16 s–23 s rRNA gene, respectively. Our optimized DPO system-based mPCR assay allowed a wide range of annealing temperature from 48 to 68 °C to efficiently amplify multi-genes followed by a nearly identical pattern with an analytical detection limit of 102–103 CFU/mL (g) for the simultaneous detection of the six target bacteria in pure cultures or artificially contaminated food matrixes. Applying the DPO system-based mPCR assay to 238 target and 83 nontarget bacterial strains revealed that only target bacterial strains were positive in this assay, indicating a high specificity. Moreover, the DPO system-based mPCR assay showed a potential diagnostic capability evaluated by testing 2419 samples collected from food, clinical and environmental sources. The DPO system-based mPCR assay may provide a useful tool for the detection of these six foodborne pathogens in laboratory diagnosis.