Rapid and sensitive detection of Yersinia pseudotuberculosis in pork using recombinase polymerase amplification-based...

Recombinase polymerase amplification (RPA) is a promising technology for amplifying nucleic acids at a constant temperature; its simple procedure, minimal equipment requirements, and fast detection times make it highly suitable for food safety applications. This study develops two rapid and sensitive assays for detecting Yersinia pseudotuberculosis in food based on RPA technology. The first assay combines RPA with a lateral flow stick (RPA-LFS) for visual readouts. The method showed high specificity against common food-borne bacteria and could detect Y. pseudotuberculosis at temperatures between 30 and 45 °C. High concentrations of templates were detected within 10 min, whereas low concentrations required 20-30 min. The limit of detection (LOD) with 95% probability by probit analysis was determined to be 206 CFU/mL. The second assay integrates RPA with real-time fluorescence detection (RT-RPA), allowing continuous monitoring of amplification products. RT-RPA detected as few as 47.5 copies/μL for pure DNA templates and 10³ CFU/mL for bacteria suspensions. Amplification occurred in 3-14 min for 10⁶-10² copies/μL of DNA template and 3-10 min for 10⁷-10³ CFU/mL of boiled bacterial suspensions. Both methods showed 100% accuracy in artificially contaminated pork samples and produced results consistent with qPCR. The RPA-LFS and RT-RPA assays constructed in this study can rapidly and accurately detect Y. pseudotuberculosis, which is crucial for food safety emergencies.