Development and Application of Recombinase Polymerase Amplification Assays for Rapid Detection of Escherichia coli O157 in Food

Escherichia coli O157 (E. coli O157) is one of the most dangerous foodborne pathogens worldwide. A convenient, sensitive, and specific method for the E. coli O157 detection is necessary. The present study developed an isothermal real-time recombinase polymerase amplification (real-time RPA) assay and an RPA combined with lateral flow strip (LFS-RPA) to detect E. coli O157 targeting the conserved region of the rfbE gene. Results of this study demonstrated that the real-time RPA was successfully conducted at the constant temperature of 39 degrees C for 20 min. Furthermore, the LFS-RPA was performed in an incubator block at 39 degrees C for 15 min, with the products visible with the naked eye within 5 min. It was found that the two RPA assays were highly specific to E. coli O157 and there were no cross-reactions with other microorganisms tested. The detection limit of E. coli O157 DNA or pure culture using LFS-RPA was 3.5 x 10(2) fg/mu L and/or 1.0 x 10(2) CFU/mL, respectively, which was 10 times higher than that of real-time PCR and real-time RPA. Moreover, the practicality of the way to discover E. coli O157 was validated with artificial contamination assay. Positive results were obtained within 6-15 min in the real-time RPA and within 15 min in the LFS-RPA, while it took approximately between 28 and 45 min in the real-time PCR. Furthermore, these assays require no sophisticated instruments, specialized technicians, and strict laboratory conditions. All of these helps to manifest that the developed RPA assays were simple, highly specific, sensitive, and rapid, and they could be employed in resource-constrained areas.

Automated summary

This study developed a simple, highly specific, sensitive, and rapid method for detecting Escherichia coli O157. The method can be conducted at a constant temperature without the need for complex equipment and strict laboratory conditions, and it does not cross-react with other microorganisms.