Application of a rapid and sensitive RPA-CRISPR/Cas12a assay for BCSP31-based Brucella detection

Brucellosis, a zoonotic disease caused by Brucella species, poses significant health risks to humans and animals. Due to the limitations of current diagnostic methods, such as serological testing and PCR, in terms of sensitivity, specificity, and speed, this study explores the potential of integrating recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system for Brucella detection. This combination leverages the strengths of both technologies for rapid, sensitive, and specific molecular diagnostics. RPA primers and CRISPR RNA (crRNA) targeting the Brucella-specific conserved sequence BCSP31 were designed, followed by optimization of the RPA-CRISPR/Cas12a system. Its performance was evaluated using genomic DNA from Brucella and non-Brucella species. The system's capabilities were assessed on clinical blood samples, demonstrating high sensitivity (detection limit of 10 copies per reaction and 16.6 attomoles for Brucella DNA) and excellent specificity. Testing on clinical samples showed strong agreement with qPCR results and an improvement over the RBT. The RPA-CRISPR/Cas12a platform represents a rapid, ultra-sensitive, and accurate method for Brucella detection and holds promise as a valuable tool for brucellosis control.