Development of Visual and Fluorescence Detection Method of Brucella by RPA ‐ CRISPR /Cas12a Assay

ABSTRACT Brucella is a significant pathogen in the livestock industry, causing Brucellosis, a zoonotic disease that leads to considerable health and economic losses in both humans and animals. Current diagnostic methods for Brucella , including culture, serological assays, and PCR/qPCR, are valuable tools but have inherent limitations. These include the requirement for BSL‐3 laboratories, trained personnel, complex procedures, expensive equipment, issues with sensitivity and specificity, and the time‐consuming nature of assays, making them unsuitable for large‐scale epidemiological screening. Therefore, there is a critical need to develop a rapid, portable, and cost‐effective diagnostic method with high specificity and sensitivity. In this study, we established a rapid, portable, reliable, and inexpensive detection method for Brucella genus identification based on RPA‐CRISPR/Cas12a technology. Specific RPA primers and crRNA sequences were designed targeting the bcsp31 gene of Brucella . Subsequently, both a fluorescence assay and a lateral flow strip (LFS) assay were developed after optimizing the conditions using the RPA‐CRISPR/Cas12a system. The limit of detection (LoD) was 1 copy/μL for RPA‐CRISPR/Cas12a‐F and 10 copies/μL for RPA‐CRISPR/Cas12a‐LFS and the entire assay was completed in less than 30 min. This method demonstrated excellent specificity in distinguishing Brucella from other closely related pathogens. Moreover, the RPA‐CRISPR/Cas12a assay showed high concordance with classical quantitative real‐time PCR when testing diverse clinical samples (blood, serum, milk, semen, vaginal secretions). Together, these findings make this method a promising tool for Brucella detection, with potential applications in both field surveillance and clinical diagnostics.