Development of Recombinase Aided Amplification (RAA)-Exo-Probe Assay for the Rapid Detection of Shiga Toxin-Producing Escherichia coli

Abstract

Abstract Background Shiga toxin-producing Escherichia coli (STEC) is a significant cause of foodborne illness causing various gastrointestinal diseases including hemolytic uremic syndrome (HUS), the most severe form, which can lead to kidney failure or even death. Objective Here, we report the development of recombinase aided amplification (RAA)-exo-probe assays targeting the stx1 and stx2 genes for the rapid detection of STEC in food samples. Methods Primers and exo-probes were designed and optimized for the detection of stx1 and stx2 using RAA technology. The optimal STEC RAA-exo-probe assays were then tested for specificity and sensitivity, and validated in both spiked and real food samples. Results These assays were found to be 100% specific to STEC strains and were also highly sensitive with a detection limit of 1.6 × 103 CFU/mL or 32 copies/reaction. Importantly, the assays were able to successfully detect STEC in spiked and real food samples (beef, mutton, and pork), with a detection limit as low as 0.35 CFU/25g in beef samples after an overnight enrichment step. Conclusions Overall, the RAA assay reactions completed within ∼20 min and were less dependent on expensive equipment, suggesting they can be easily adopted for in-field testing requiring only a fluorescent reader. Highlights As such, we have developed two rapid, sensitive, and specific assays that can be used for the routine monitoring of STEC contamination in food samples, particularly in the field or in poorly equipped labs.