Detection of viable toxigenic Vibrio cholerae from environmental water sources by direct cell duplex PCR assay

Environmental monitoring is important to enable effective resource management and public health protection as well as rapid and accurate identification of Vibrio cholerae in drinking-water sources. Traditional methods employed in identification are laborious, time-consuming and practically not viable for screening of a large number of samples. In this study, a direct cell duplex PCR assay for the detection of viable toxigenic V. cholerae in environmental water samples was developed. In the PCR assay, two gene sequences were amplified together, one of outer membrane protein (ompW), which is species-specific and another of cholera toxin (ctxAB). The detection limit of duplex PCR was 5 x 10(4) V. cholerae/reaction. Different environmental water samples were artificially spiked with V. cholerae O1 cells and filtered through a 0.22 mu m membrane, and the filters enriched in alkaline peptone water for 6 h and then used directly in the duplex PCR assay. The PCR procedure coupled with enrichment could detect as few as 1.2 c. f.u./ml in ground water, 1.2 x 10(2) c.f.u. ml(-1) in sewer water and 1.2 x 10(3) c.f.u. ml(-1) in tap water. The assay was successfully applied directly for screening of environmental potable water samples collected from a cholera-affected area. The proposed method is simple and can be used for environmental monitoring of toxigenic as well as non-toxigenic V. cholerae.