Development of a rapid and sensitive RPA-CRISPR/Cas12a-based assay for the detection of Brucella melitensis.

Brucellosis, a zoonotic disease caused by Brucella species, presents significant public health challenges due to its complex diagnosis and the limited availability of rapid detection methods. To address these challenges, we developed a novel detection method that integrates recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system, enabling dual readout through fluorescence (FL) and lateral flow strip (LFS) detection. The RPA-CRISPR/Cas12a-FL assay demonstrated an impressive detection limit of 1 copy/μL, which is 10 times more sensitive than quantitative polymerase chain reaction, while the RPA-CRISPR/Cas12a-LFS method achieved a detection limit of 10 copies/μL, comparable to nested PCR. Specificity testing confirmed the robustness of the assay, as it produced strong signals exclusively for Brucella without cross-reactivity with other bacterial species. Clinical validation using serum samples from 24 confirmed brucellosis patients and six healthy controls demonstrated a 100% concordance with serological results, underscoring the reliability of this method for clinical applications. This assay provides a rapid, sensitive, and specific tool for Brucella detection, suitable for both laboratory and field settings, and holds significant potential for enhancing the diagnosis and control of brucellosis.IMPORTANCEBrucellosis is a significant zoonotic disease, and rapid and accurate diagnosis is crucial for its treatment and control. To address this need, we developed a novel detection method that combines recombinant enzyme polymerase amplification with a CRISPR/Cas12a system, achieving dual readout through fluorescence and lateral flow strips. The test demonstrates excellent sensitivity and specificity, with clinical validation confirming complete concordance with serological results. This approach offers a fast, reliable, and field-deployable solution for brucellosis diagnosis, significantly enhancing disease management and public health outcomes.