Development of a One-Tube RPA-CRISPR/Cas12a assay for the detection of Brucella

Abstract Background Brucellosis, a zoonotic disease caused by Brucella species, poses significant fhealth risks to humans and animals. Current diagnostic methods suffer from limitations such as long turnaround times, reliance on specialized equipment, or insufficient sensitivity and specificity. Results A one-tube RPA-CRISPR/Cas12a assay targeting the highly conserved aroB gene for rapid, sensitive, and specific detection of Brucella was developed. The optimal crRNA and RPA primer pairs were screened, and the reaction conditions including temperature, Cas12a, crRNA, and ssDNA reporter concentrations were systematically optimized. The assay was performed in a closed single tube to minimize aerosol contamination and simplify operation, allowing the entire detection process to be completed within 50 min. Analytical evaluation demonstrated that the method achieved a limit of detection (LOD) of 2.3 copies/μL (1.15 copies/reaction) and high specificity, with no cross-reactivity against Escherichia coli , Mycobacterium tuberculosis , Salmonella , Chlamydia abortus , or Coxiella burnetii . When tested on 30 simulated clinical sheep serum samples, the assay yielded 100% agreement with the national standard qPCR method (GB/T 18646–2025), providing technical feasibility evidence for its potential clinical application. Conclusions Collectively, this one-tube RPA-CRISPR/Cas12a assay can provide a technical reference for the rapid diagnosis of brucellosis. While the assay demonstrates excellent analytical performance, comprehensive clinical diagnostic performance assessment requires further systematic verification using large batches of authentic clinical samples.