Development of O-serogroup specific PCR assay for detection and identification of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a human pathogen that is widely disseminated in estuarine, marine and coastal environments throughout the world, and is recognized as the leading cause of food-borne illness worldwide. V. parahaemolyticus infections have been characterized by causal associations with multiple, diverse serotypes. To date, 13 O-serogroups have been recognized in V. parahaemolyticus, although only the O-serogroup genetic determinants (OGDs) of serogroups O3 and O4 have been sequenced. In this study, the OGDs of the remaining 11 serogroups were identified. A PCR assay based on O-serogroup specific genes was developed for the identification and detection of all 13 V. parahaemolyticus O-serogroups and tested against 41 target strains and 21 strains of other bacterial species. A double-blind test including 105 environmental specimens was also performed. The developed method was shown to distinguish all V. parahaemolyticus O-serogroups effectively with the only exception of O3 and O13. The method was found to be highly specific and reproducible, with detection sensitivity of 1ng of genomic DNA, and it was demonstrated that V. parahaemolyticus at the level of 10(4)CFU/ml in mock water specimens and the enrichment culture of samples inoculated with at the level of 1CFU/ml were detected. As few as 2 to 18CFU (initial inoculum) of V. parahaemolyticus were detectable in a 1g oyster sample after enrichment using this PCR method. The molecular protocol developed in this study for identification of all V. parahaemolyticus serogroups is therefore suitable for rapid detection and identification of V. parahaemolyticus pathogens from clinical and environmental samples, with the potential for application in epidemiologic investigations and other food safety applications.