[Development and application of a triplex basic- recombinase polymerase amplification method to detect Brucella,...

To combat three major pathogens-Brucella spp.,Babesia bovis, and Pasteurella multocida-threatening cattle health, we developed a rapid and accurate triplex basic-recombinase polymerase amplification (Basic-RPA) detection system. Targeting bcsp31, rap-1, and kmt1, we designed and screened specific RPA primers and optimized the reaction temperature, time, and primer concentration. The best reaction conditions were 38 ℃ for 25 min, with primers at 10 μmol/L in a 1:1:1 ratio. We then used reference and non-target strains to evaluate the specificity of the established method and serial DNA dilutions to evaluate the method sensitivity and repeatability. Subsequently, we applied the method to detection of 640 samples collected from cattle farms, slaughterhouses, supermarkets, and wet markets. The limit of detection of this method for all the three pathogens reached 10³ CFU/mL (copies/mL). The method showed high specificity, excellent repeatability, and 100% consistency with PCR results. Among the samples, Brucella spp., B. bovis, and P. multocida were detected at rates of 0.31%, 2.34%, and 1.25%, respectively, indicating different patterns of pathogen distribution across production stages. In conclusion, our triplex Basic-RPA method is simple, sensitive, and highly specific. It provides an efficient molecular tool for rapid detection and surveillance of bovine pathogens along the farm-to-market chain and lays the groundwork for developing advanced RPA-based technologies such as recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) and recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats, (RPA-CRISPR).