Detection of viable toxigenic Vibrio cholerae and virulent Shigella spp. in environmental waters by pit-stop seminested polymerase chain reaction assays

A rapid and sensitive assay was developed for the detection of low numbers of viable Vibrio cholerae and Shigella spp. cells in environmental and drinking water samples. Water samples were filtered, and the filters were enriched in a non-selective medium. The enrichment cultures were prepared for polymerase chain reactions (PCR) by a rapid and simple DNA extraction procedure consisting of boiling. Seminested PCR, based on specific amplification of the cholera toxin operon of V. cholerae and the invasion plasmid antigen gene (ipaH) of virulent Shigella spp., was performed and the PCR products were visualised by agarose gel electrophoresis. The assay allowed the detection of as few as 1 cfu/100 ml of V. cholerae and 8 cfu/100 ml of Shigella cells. A comparison of the PCR method and culturing methods by using environmental water samples showed that the PCR method has a higher level of sensitivity than culturing methods. As an application of the PCR detection protocol, environmental water samples were screened for the presence of V. cholerae and Shigella spp. Positive amplifications resulted from V. cholerae and Shigella species in environmental samples. The results obtained indicate that the described seminested PCR has the advantage of a rapid turn-around time and fulfils the requirements of sensitivity for use in an environmental laboratory.