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 degrees C to efficiently amplify multi-genes followed by a nearly identical pattern with an analytical detection limit of 10(2)-10(3) 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.