Development of a One-Pot MIRA-CRISPR/Cas12b Fluorescence Biosensing Platform for Rapid Detection of Brucella canis

Brucellosis is an important zoonotic disease worldwide, and Brucella canis (B. canis) is an emerging concern in companion animal health. Rapid and reliable detection of B. canis in dogs is essential for disease control. Herein, we developed a one-pot MIRA CRISPR/Cas12b fluorescence biosensing platform targeting the conserved omp31 gene. The platform integrates MIRA mediated isothermal amplification with CRISPR/Cas12b-based target recognition and collateral cleavage. After optimization, the assay achieved a detection limit of 5 copies/reaction within 20 min, comparable to qPCR. No cross reactivity was observed with seven common canine pathogens. Among 128 clinical blood samples, the biosensor showed complete agreement with qPCR, achieving 100% sensitivity and 100% specificity. These findings demonstrate that the biosensor provides a rapid, sensitive, and specific approach for B. canis detection, with potential for decentralized veterinary testing through its endpoint visual fluorescence readout and applications in One Health surveillance.