Brucellosis remains an important zoonotic disease for livestock production and public health, creating a need for rapid molecular screening methods with limited equipment requirements. We developed a visual recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay targeting a 642-bp region of the Brucella omp25 gene. Comparative sequence analysis was used to assess target conservation, after which four RPA primer pairs and two CRISPR RNAs (crRNAs) were experimentally screened. RPA3-crRNA1 produced the strongest fluorescence response and was used for assay optimization. The final Cas12a reaction contained 250 nM Cas12a, 200 nM crRNA, and 200 nM single-stranded DNA fluorescent reporter. RPA amplification and Cas12a detection required 20 min each, and positive reactions produced visible yellow-green fluorescence under 470-nm blue light. No cross-reactivity was observed with the non-target pathogens included in the experimental panel. In silico comparisons with selected phylogenetically related taxa identified sequence differences within the final RPA3 primer-binding sites and crRNA1 recognition region, supporting a low predicted risk of sequence-based cross-reactivity for the taxa examined. Using purified plasmid DNA, the lowest DNA concentration detected under the experimental conditions tested was 2.72 copies/μL of input template, corresponding to an estimated 13.6 copies per RPA reaction. Among 96 bovine nasal swab samples collected during routine surveillance, agreement analysis against the qPCR comparator showed concordant results for 93 of 96 samples, with positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) of 100.00, 93.75, and 96.88%, respectively; three qPCR-negative samples showed weak RPA-CRISPR/Cas12a fluorescence and could not be independently resolved. Because the target region is conserved in the vaccine strains A19, S19, and RB51, the assay is intended for preliminary screening of brucellosis-associated Brucella DNA rather than species identification or differentiation of vaccine-derived and wild-type DNA. Broader validation with additional specimen types and specificity panels is required before routine diagnostic application.
