Abstract
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 °C for 20 min. Furthermore, the LFS-RPA was performed in an incubator block at 39 °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 × 102 fg/μL and/or 1.0 × 102 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.
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Data availability
The dataset analyzed during the current study is available from the corresponding author on reasonable request.
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Funding
This work was supported by the program of Traditional Chinese Medicine Scientific Research foundation in Hebei Administration of Traditional Chinese Medicine (2020142 and 2019091, Hebei, China), the Natural Science Foundation of Hebei Province (C2020423052), the Project of Excellent Young Teacher Fundamental Research (YQ2019003), and Doctoral Foundation (BSZ2019009) of Hebei University of Chinese Medicine.
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YYG and JCW designed and conducted the experiment. LWZ, MNC, XXS, and YYW performed the experiments and analyzed the data. YYG drafted the manuscript. All authors read, revised, and approved the final manuscript.
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Zhao, L., Wang, J., Chen, M. et al. Development and Application of Recombinase Polymerase Amplification Assays for Rapid Detection of Escherichia coli O157 in Food. Food Anal. Methods 15, 1843–1850 (2022). https://doi.org/10.1007/s12161-022-02250-1
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DOI: https://doi.org/10.1007/s12161-022-02250-1