Recombinase polymerase amplification for fast, selective, DNA-based detection of faecal indicator Escherichia coli

The bacterium Escherichia coli is commonly associated with the presence of faecal contamination in environmental samples, and is therefore subject to statutory surveillance. This is normally done using a culture-based methodology, which can be slow and laborious. Nucleic acid amplification for the detection of E. coli DNA sequences is a significantly more rapid approach, suited for applications in the field such as a point of sample analysis, and to provide an early warning of contamination. An existing, high integrity qPCR method to detect the E. coli ybbW gene, which requires almost an hour to detect low quantities of the target, was compared with a novel, isothermal RPA method, targeting the same sequence but achieving the result within a few minutes. The RPA technique demonstrated equivalent inclusivity and selectivity, and was able to detect DNA extracted from 100% of 99 E. coli strains, and exclude 100% of 30 non-target bacterial species. The limit of detection of the RPA assay was at least 100 target sequence copies. The high speed and simple, isothermal amplification chemistry may indicate that RPA is a more suitable methodology for on-site E. coli monitoring than an existing qPCR technique.