Simultaneous detection of Vibrio cholerae, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio vulnificus in seafood using dual priming oligonucleotide (DPO) system-based multiplex PCR assay

- A DPO-based mPCR assay for four pathogenic Vibrio species in seafood was developed.
- The DPO-based mPCR assay allowed a wide annealing temperature range.
- The DPO-based mPCR assay was more specific than conventional PCR assay.
- The assay has been validated in food, clinical samples and foodborne outbreaks.
- The assay is reliable tool for detecting the four Vibrio species in lab diagnosis.

In this study, a rapid and reliable multiplex PCR assay for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus in seafood was developed using the dual priming oligonucleotide (DPO) system. Species-specific DPO primers were designed targeting the mdh, vvhA, colH and toxR genes for the discrimination of V. cholerae, V. vulnificus, V. alginolyticus and V. parahaemolyticus, respectively. Compared to conventional PCR assay, the DPO system-based multiplex PCR assay allowed a wider annealing temperature at 48 °C-68 °C to effectively amplify target genes followed an analytical detection limit of <1.5 × 102 CFU/mL (or g) for each Vibrio species in pure cultures or artificially contaminated food matrix. A total of 396 bacterial strains including 209 targets and 187 other bacterial strains were used to test the specificity of the DPO system-based multiplex PCR assay, and results showed that specific PCR product was only observed in target bacterial strains without nonspecific priming. Practical application of the assay indicated that target Vibrio species in seafood, clinical samples and foodborne outbreaks can be accurately detected. This DPO system-based multiplex PCR assay developed in this study would be a powerful tool for the rapid and reliable detection of the target Vibrio species.