Detection of toxigenic Vibrio cholerae, V. parahaemolyticus and V. vulnificus in oyster by multiplex-PCR with internal amplification control

Abstract

This study developed a multiplex-polymerase chain reaction (m-PCR) method that targets the toxin genes, vvhA and vvp for Vibrio vulnificus, tdh for V. parahaemolyticus, ctx for V. cholera, to detect these three species simultaneously. A chimeric DNA consisting of a fragment of the green fluorescent protein gene (gfp) flanked by sequences of the vvhA primers was used as the internal amplification control (IAC). In the presence of these three Vibrio species, amplicons of IAC (753 bp), vvhA (505 bp), vvp (383 bp), tdh (256 bp) and ctx (167 bp) could be simultaneously detected. The accuracy of the m-PCR approach was evaluated with a Kappa index of 0.96 using 28 strains of V. vulnificus, 18 V. parahaemolyticus, 14 V. cholerae and 40 other Vibrio species and non-Vibrio strains. As little as 101 CFU/mL of these Vibrio species in spiked oyster homogenate could be detected by this m-PCR method following enrichment at 37°C for 8 h. This method can be adopted for the rapid detection of the toxigenic strains of Vibrio species in seafood.