Triplex reverse transcription-PCR for detecting viable toxigenic Vibrio cholerae in water samples in Thailand.

Abstract. Detection of toxigenic Vibrio cholerae O1/O139 in aquatic environment is difficult to achieve using the culture method. For direct detection of viable toxigenic V. cholerae in aquatic environment, we developed a triplex reverse transcription (RT)-PCR, targeting genes for the outer membrane protein (ompW), cholera toxin A (ctxA) and toxin-coregulated pilli (tcpA) and compared the assay with the culture method. After enrichment of the bacteria-containing filters in alkaline peptone water for 6 hours, the sensitivity of triplex RT-PCR for detecting V. cholerae was 7 cfu/ml. Of the 80 environmental water samples collected from various regions in Northeast Thailand, triplex RT-PCR detected 15 toxigenic and 20 non-toxigenic V. cholerae, whereas the culture method detected only 3 toxigenic V. cholerae--containing water samples. These results show that this triplex RT-PCR method could be used as an alternative tool for rapid and sensitive identification of viable toxigenic V cholerae in environmental water samples.