Development of a Visual RPA-CRISPR/Cas12a Assay Targeting Bcsp31 for Rapid Detection of Brucella spp

To establish a rapid nucleic acid detection method for field screening of Brucella spp., a visual assay based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a was developed. Four pairs of RPA primers and two crRNAs were designed according to the conserved Brucella bcsp31 sequence. The optimal RPA primer-crRNA combination was screened using agarose gel electrophoresis and fluorescence readout, and the concentrations of Cas12a protein, crRNA, and ssDNA fluorescent reporter were optimized. RPA primer pair 2 showed the best amplification performance, and RPA2-crRNA1 was selected as the optimal combination for the assay. The final reaction conditions were 200 nM Cas12a protein, 200 nM crRNA, and 250 nM ssDNA fluorescent reporter. The limit of detection of the established method was 2.47 × 10⁰ copies/μL. No cross-reactivity was observed with Mycoplasma capricolum subsp. capripneumoniae, Chlamydia abortus, Chlamydia psittaci, or Salmonella enterica serovar Typhimurium. Among 56 clinical sheep nasal swab samples collected during routine surveillance, qPCR identified 22 positive and 34 negative samples, while the RPA-CRISPR/Cas12a assay identified 20 positive and 36 negative samples. The positive agreement, negative agreement, overall agreement, positive predictive value, negative predictive value, and Cohen's kappa value were 90.91%, 100.00%, 96.43%, 100.00%, 94.44%, and 0.924, respectively. These findings provide preliminary evidence that the developed bcsp31-targeted RPA-CRISPR/Cas12a assay is rapid and visually interpretable and may be useful for preliminary Brucella spp. screening in routine veterinary surveillance settings. Larger-scale validation using different sample types and samples from different regions is still required.