Development and Evaluation of Recombinase-Aided Amplification Assay for Rapidly Detecting Escherichia coli in Urine

Escherichia coli is the main pathogen of urinary tract infection (UTI). Early and rapid detection for UTI pathogens is still limited. The aim of this study was to develop and evaluate a recombinase-aided amplifica-tion (RAA) assay for rapidly detecting Escherichia coli in urine.

RAA specific primers and probes were designed based on the conserved region of E. coli chuA gene. The optimal primers and probe combinations were screened and identified using a real-time fluorescent RAA assay. The specificity was assessed by detecting E. coli and other none-E. coli uropathogens. The limit of detection (LOD) was determined through serial dilution. One hundred thirty-five midstream urine samples were used to evaluate the sensitivity, specificity, and accuracy. RAA and urine culture were performed in parallel. Urine culture is still the gold standard.

The optimal combination of 2 primers with 1 probe was determined based on the real-time fluorescence RAA curves. The LOD of this method is 103 cfu/mL. No cross-reactivity was observed with 12 other uropathogens that are commonly encountered in UTI. Among the 135 urine culture results, 50 of them were E. coli. 75 of them were non-E. coli. The remaining 10 were culture negative. They were detected by both RAA assay and urine culture in parallel. The sensitivity, specificity and accuracy of RAA assay were 94.00%, 91.76%, and 92.59%, respectively.

The real-time fluorescent RAA assay for urinary E.coli established in this study is a highly sensitive, specific and cost-effective method. It can be utilized for rapid detection of urinary E.coli within 30 minutes.