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Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli.A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxonomic specificity and sensitivity of qPCR assays targeting the rodA gene (rodA984) and two regions of the multi-copy 23S ribosomal RNA gene (EC23S and EC23S857). Experimental analyses of 28 culture collection strains representing E. coli and 21 related non-target species indicated that the uidA405 and rodA984 assays were both 100% specific for E. coli while the EC23S assay was only 29% specific. The EC23S857 assay was only 95% specific due to detection of E. fergusonii. The uidA405, rodA984, EC23S and EC23S857 assays were 85%, 85%, 100% and 86% sensitive, respectively, in detecting 175 presumptive E. coli culture isolates from fresh, marine and waste water samples. In analyses of DNA extracts from 32 fresh, marine and waste water samples, the rodA984, EC23S and EC23S857 assays detected mean densities of target sequences at ratios of approximately 1 : 1, 243 : 1 and 6 : 1 compared with the mean densities detected by the uidA405 assay.The EC23S assay was less specific for E. coli, whereas the rodA984 and EC23S857 assay taxonomic specificities and sensitivities were similar to those of the uidA405 gene assay.The EC23S857 assay has a lower limit of detection for E. coli cells than the uidA405 and rodA984 assays due to its multi-copy gene target and therefore provides greater analytical sensitivity in monitoring for these faecal pollution indicators in environmental waters by qPCR methods.