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. par-
ahaemolyticus, respectively. Compared to conventional PCR assay, the DPO system-based multiplex PCR
assay allowed a wider annealing temperature at 48 Ce68 C to effectively amplify target genes followed
an analytical detection limit of