Use of a high resolution melt real-time polymerase chain reaction (PCR) assay for the environmental monitoring of Vibrio cholerae

Abstract

A real-time polymerase chain reaction (PCR) assay utilizing high resolution melt (HRM) curve analysis was developed and tested for the monitoring of Vibrio cholerae in water samples. The assay utilized previously published primers that are specific to regions of the V. cholerae ompW and ctxAB genes, allowing it to differentiate between toxigenic and non-toxigenic strains. The ompW and ctxAB primers amplify target regions of 588 and 564 bp in length (respectively) and the amplicons could be accurately identified using HRM curve analysis. High resolution melt curve analysis provided additional accuracy for the determination of amplicon melting temperatures, and allowed amplification of the two targets in a multiplex reaction. Two laboratories employed the assay to analyse 178 water samples obtained from diverse environmental water sources, for the presence of V. cholerae. The assay was found to be a rapid, highly accurate, sensitive and cost effective method for the detection and distinction between toxigenic and non-toxigenic V. cholerae strains in water.