Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW

The distribution of genes for an outer membrane protein (OmpW) and a regula tory protein (ToxR) in Vibrio cholerae and other organisms was studied usin g respective primers and probes. PCR amplification results showed that all (100%) of the 254 V. cholerae strains tested were positive for ompW and 229 (similar to 98%) of 233 were positive for toxR. None of the 40 strains bel onging to other Vibrio species produced amplicons with either ompW- or toxR -specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay. These studies were extended with r epresentative number of strains using ompW- and toxR-specific probes in DNA dot blot assay. While the V. cholerae strains reacted with ompW probe, onl y one (V. mimicus) out of 60 other bacterial strains tested showed weak rec ognition. In contrast, several strains belonging to other Vibrio species (e .g., V. mimicus, V. splendidus, V. alginolyticus, V. fluvialis, V. proteoly ticus, V. aestuarianus, V., lsalmonidida, V. furnissii, and V. parahaemolyt icus) showed weak to strong reactivity to the toxR probe. Restriction fragm ent length polymorphism analysis and nucleotide sequence data revealed that the ompW sequence is highly conserved among V. cholerae strains belonging to different biotypes and/or serogroups. All of these results suggest that the ompW gene can be targeted for the species-specific identification of V. cholerae strains. The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplifi cation of ompW and ctxA genes which should be of considerable value in the screening of both toxigenic and nontoxigenic V. cholerae strains of clinica l as well as environmental origin.