Development and implementation of a rapid real-time polymerase chain reaction assay for the detection of toxigenic <i>Vibrio cholerae</i> in water

Abstract

Assays which use real-time polymerase chain reaction (PCR) technology can be developed for the rapid identification of genetic sequences carried by waterborne pathogens. Rand Water has established facilities within which a selection of PCR assays will be developed. This paper reports on the optimisation and validation of the first assay to be implemented. This assay facilitates the detection of the ctxA gene of toxigenic Vibrio cholerae (V. cholerae) strains. The assay also includes an internal process control in the form of an Escherichia coli (E. coli) strain carrying a single genomic copy of the gfp gene from Aequorea victoria. Establishment of the assay required the selection of suitable PCR primers and probes for both the ctxA and gfp genes. This was followed by an optimisation phase where ideal PCR cycling conditions and primer/probe concentrations were established. A validation phase established the performance parameters of the assay. Parameters assessed included: limit of detection, sensitivity, specificity, reproducibility and robustness. The validation was conducted using potable water, surface water and sewage effluent matrices. The process has resulted in the establishment of a robust assay for the detection of toxigenic V. cholerae strains within 24 hours after samples are received.Keywords: real-time PCR, Vibrio cholerae O1, ctxA gene, validation