Absolute quantification of Vibrio parahaemolyticus by multiplex droplet digital PCR for simultaneous detection of tlh, tdh and ureR based on single intact cell

Vibrio parahaemolyticus, a marine food-borne pathogen, has been proved to be a significant cause of human gastrointestinal disorders worldwide. In this study, a method of multiplex droplet digital PCR (ddPCR) based on primer and probe sequences of the tlh, tdh and ureR genes were developed and evaluated for the reliable quantification of V. parahaemolyticus cells in seafoods. The specificities of all primers and probes used in this study were validated on three standard strains of V. parahaemolyticus, 10 strains of Vibrio spp., and 22 strains of other bacteria by ddPCR and quantitative PCR (qPCR). Then the ddPCR system, primers, probe concentration and amplification procedures were optimized, and the templates with cell and genomic DNA (gDNA) were compared. The results showed that using cell as template could benefit for multiplex ddPCR, which performed higher linkage among three genes. This method improved sensitivity, specificity, accuracy, convenience, and reproducibility for the detection of V. parahaemolyticus, and the limit of detection (LOD) was 15 CFU/mL. In addition, the applicability of this method was compared with plate count and qPCR, and then verified to detect artificially contaminated seafood samples containing different concentrations of V. parahaemolyticus. The results indicated that the established method is stable, accurate, sensitive, range-wide, and has the potential to detect the three different genes of V. parahaemolyticus in food samples.