Detection of sewage-derived escherichia coli in a rural stream using multiplex PCR and automated DNA detection

A randomly amplified polymorphic DNA (RAPD) marker, localised to the glycine decarboxylase gene (gcvP) of Escherichia coli, has shown promise for use as a molecular marker for the identification E. coli isolates of human origin. Characterisation of the RAPD polymorphism has enabled development of target-specific primers for direct PCR detection of E. coli of human origin. A field trial was undertaken to investigate the suitability of the marker for detecting E. coli derived from sewage effluent discharged to a rural stream which already contained a substantial background level E. coli of animal origin. Multiplex PCR was performed on E. coli isolates from each sample site using the marker-specific primers in conjunction with primers directed towards a region of the β-glucuronidase (gusA) gene which served as an internal PCR control. Marker-positive strains were detected in raw sewage, treated effluent, stream water collected from immediately downstream of the effluent discharge and from a small tributary which ran down one side of the treatment plant. However, no marker-positive isolates were detected upstream of the treatment plant, despite the relatively high faecal coliform levels (mean = 3.5×103cfu/100ml) recorded at these sites. Further investigations on the side tributary revealed a possible entry point for sewage from the treatment system. Thus the PCR-based approach described here shows promise for the detection of human faecal contamination in rural environments and may be of use for other environmental applications such as the detection of septic tank leachate in rural catchments.