Single multiplex polymerase chain reaction for environmental surveillance of toxigenic—Pathogenic O1 and Non-O1vibrio cholerae

A multiplex PCR assay was developed for the detection of toxigenic and pathogenicV. cholerae from direct water sources using specific primers targeting diverse genes,viz. outer membrane protein (ompW), cholera toxin (ctxB), ORF specific for O1 (rfbG), zonula occludens (zot) and toxin co-regulated pilus (tcpB); among these genes,ompW acts as internal control forV. cholerae, thectx gene as a marker for toxigenicity andtcp for pathogenicity. The sensitivity of multiplex PCR was 5 × 104V. cholerae cells per reaction. The procedure was simplified as direct bacterial cells were used as template and there was no need for DNA extraction. The assay was specific as no amplification occurred with the other bacteria used. ToxigenicV. cholerae were artificially spiked in different water samples, filtered through a 0.45 µm membrane, and the filters containing bacteria were enriched in APW for 6 h. PCR following filtration and enrichment could detect as little as 8V. cholerae cells per mL in different spiked water samples. Various environmental potable water samples were screened for the presence ofV. cholerae using this assay procedure. The proposed method is rapid, sensitive and specific for environmental surveillance for the presence of toxigenicpathogenic and nonpathogenicV. cholerae.