Establishment of a rapid method for the detection of Brucella canis based on recombinase-mediated thermostable nucleic acid amplification technology

To establish a rapid detection method for canine using recombinase-aided amplification (RAA) technology. The outer membrane protein 25 gene fragment (Omp25) of canis was targeted. Primers and fluorescent probes were designed and synthesized, and recombinant plasmids were constructed as standards. The RAA assay was optimized by screening primers and establishing a fluorescent reaction system. Sensitivity was analyzed using plasmid standards with varying copy numbers. Specificity was tested using genomes from canis, suis, melitensis, abortus, spp. Reproducibility was evaluated using plasmid standards from the same and different batches. The optimized RAA system used primers bOmp25-F2/bOmp25-R2 and probe bOmp25-P, with a constant reaction temperature of 39°C for 15 minutes. The detection sensitivity was 1 copy/μL. No cross-reaction was observed with other species or pathogenic bacteria, indicating high specificity. Intra-batch variability was below 1.00%, and inter-batch variability was below 2.00%. The positive detection coincidence rate of RAA was significantly higher than that of commercial real-time fluorescence quantitative PCR (100% VS 86.96%, P