Implementation of a process internal control to monitor the performance of real-time PCR assays for the detection of Vibrio cholerae in water

The process control utilised in this study was an Escherichia coli (E. coli-GFP) strain carrying a chromosomally integrated copy of the green fluorescent protein (gfp) gene of the jelly fish Aequorea victoria. Application of the process control involved spiking samples with the E. coli-GFP containing cells and detecting gfp using real-time polymerase chain reaction (PCR). The process control was implemented in the context of two multiplex real-time PCR assays. The first multiplex targeted the ctxA, hlyA and gfp genes, while the second targeted the gfp, O1-rfb, and O139-rfb loci. The detection limits for both the multiplex real-time PCR assays were 20 CFU (colony forming units) per reaction. This method was evaluated using spiked and unspiked Alkaline Peptone Water (APW) enrichments of sewage, dam, catchment and tap water. The internal process control showed no substantial inhibition in the APW enriched water samples, assuring the integrity of negative results. The results from this study showed that the use of the E. coli-GFP strain as a process internal control could provide quality assurance and increase the reliable interpretation of real-time PCR results.