Multiplex PCR for the Identification of Pathogenic in Plant Based on Novel Specific Targets Revealed by Pan-Genome Analysis.

spp. is an important foodborne disease agent, often found in the fresh mushroom () and its production environment. The aim of this study was to develop multiplex PCR for rapid identification of and , and nonpathogenic in plants. Pan-genome analysis was first used to identify five novel -specific targets: one for the genus, one for , and three for . Primers for the novel targets were highly specific in individual reactions. The detection limits were 10-10 CFU/mL, meeting the requirements of molecular detection. A mPCR assay for the identification of pathogenic , with primers targeting the novel genes specific for genus (), (), and () was then designed. The assay specificity was robustly verified by analyzing nonpathogenic and non- spp. strains. The determined detection limits were 2.0 × 10 CFU/mL for s and 3.4 × 10 CFU/mL for , for pure culture analysis. Further, the assay detected 7.6 × 10 to 7.6 × 10 CFU/10 g of pathogenic spiked into samples following 4-12 h enrichment. The assay feasibility was evaluated by comparing with a traditional culture-based method, by analyzing 129 samples collected from different plants. The prevalence of spp. and was 58.1% and 41.1%, respectively. The calculated κ factors for spp., , and were 0.97, 0.97, and 1, respectively. The results of the novel mPCR assay were highly consistent with those of the culture-based method. The new assay thus will allow rapid, specific, and accurate detection and monitoring of pathogenic in food and its production environment.