Development of a multiplex PCR assay for the detection of major virulence genes in Vibrio cholerae including non-O1 and non-O139 serogroups.

Vibrio cholerae strains producing cholera toxin (CT) and toxin co-regulated pilus (TCP) and belonging to O1 and O139 serogroups are responsible for cholera. However, non-CT producing V. cholerae from non-O1/non-O139 serogroups have been increasingly isolated from diarrheal stools and extra-intestinal infections. In this study, we have developed a multiplex PCR for the simultaneous detection of heat-stable enterotoxin (stn), type three-secretion system (vopF), and cholix toxin (chxA), along with CT (ctx) in V. cholerae strains. As other species from genus Vibrio carries homologous virulence genes, V. cholerae specific ompW was also included to differentiate V. cholerae from other vibrios. This assay was 100% specific and sensitive, and could detect homologous virulence genes like ctxA in V. mimicus and vopF in V. parahaemolyticus. This multiplex PCR assay, which can detect four major virulence genes in V. cholerae, is novel and important for epidemiologic and environmental surveillance of pathogenic V. cholerae.