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. Significance and impact of the study: In this study, recombinase polymerase amplification (RPA) is presented as a fast, and highly selective method for the detection Escherichia coli DNA from diverse environmental strains. A novel RPA assay was compared with an existing, high performance qPCR, and demonstrated an equivalent inclusivity and specificity for the target species, with a significantly reduced analysis time. The RPA could be used to amplify and detect E. coli DNA in fewer than 3 min. The speed, selectivity and isothermal low temperature requirements of the RPA technique make it well‐suited for on‐site water quality testing.