Abstract: Recombinase polymerase amplification (RPA) combined with a centrifugal compact disc microfluidic chip system was used to develop a method for the detection of Shiga toxin-producing Escherichia coli (STEC). Specific primers and probes targeting the stx1 and stx2 genes were designed and evaluated. The efficiency of RPA was verified using 19 STEC strains including 9 stx gene subtypes and 21 non-STEC strains. The fluorescence RPA-integrating microfluidic chip was evaluated using artificially contaminated beef samples. The inclusivity and exclusivity of fluorescence RPA were 100% when compared to the real-time polymerase chain reaction (real-time PCR) from the Microbiology Laboratory Guidebook of USDA, namely the United States Department of Agriculture. The fluorescence RPA microfluidic chip method could conduct 32 reactions simultaneously within 20 min with a sensitivity of 9.5 × 103 CFU/mL for STEC strains. The STEC strains could be detected in artificially contaminated beef samples at an inoculation level of 1 CFU/25 g after enrichment culture by the GB 4789.6-2016 Food Microbiological Examination-Diarrheagenic Escherichia coli. The relative trueness and the relative detection level of the method were both 100%. The fluorescence RPA microfluidic chip method can detect 9 stx gene subtypes of STEC with simple operation and fast response, and it is suitable for rapid and high-throughput detection of STEC.

Key words: recombinase polymerase amplification; microfluidic chip; Shiga toxin-producing Escherichia coli; stx gene; rapid detection