Novel PCR-based genotyping method,using genomic variability between repetitive sequences of toxigenic Vibrio cholerae O1 El Tor and O139

A novel genotyping method for toxigenic Vibrio cholerae O1 El Tor and O139 was developed. The method was designed to amplify DNA sequences “sandwiched“ between any given pair of repetitive sequences, “V. cholera repeats (VCR)”, in highly polymorphic “integron island” of ca. 125 kb in the small chromosome of toxigenic V. cholerae so that the resultant PCR amplicons would present with a strain-specific electrophoretic pattern. The VCR-targeted PCR assay (VCR-PCR) for 37 strains of toxigenic V. cholerae O1 El Tor and O139 revealed that the O1 strains isolated before 1990 showed distinct clonality whereas those isolated after 1990 could be separated into two clones, one consisting of strains isolated from South American countries and another of those from other countries. By contrast, O139 strains were genotypically homogenous regardless of the geographic origin or time of isolation. VCR-PCR therefore would be a robust but rapid method for genotypic differentiation of toxigenic V. cholerae O1 El Tor and O139 strains and to recognize strains with epidemic potential.