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Published on: April 25, 2025
Development of Visual and Fluorescence Detection Method of Brucella by RPA-CRISPR/Cas12a Assay
Hafiz Muhammad Hamza Rasool1, Xiaowei Gong1, Youshun Jin1
1State Key Laboratory of Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences, Lanzhou, China.
A new RPA-CRISPR/Cas12a assay offers rapid, portable, and cost-effective detection of Brucella bacteria. This method provides high specificity and sensitivity, outperforming traditional diagnostics for Brucellosis surveillance.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Zoonotic Disease Research
Background:
- Brucellosis, caused by Brucella, results in significant economic and health losses globally.
- Current Brucella diagnostics (culture, serology, PCR) have limitations like BSL-3 requirements, complexity, and time, hindering epidemiological screening.
- A need exists for rapid, portable, sensitive, and specific Brucella detection methods.
Purpose of the Study:
- To develop and validate a novel, rapid, portable, and cost-effective diagnostic method for Brucella genus identification.
- To establish a detection system based on Recombinase Polymerase Amplification (RPA) combined with CRISPR/Cas12a technology.
- To optimize and assess the performance of the RPA-CRISPR/Cas12a assay for Brucella detection.
Main Methods:
- Designed specific RPA primers and CRISPR/Cas12a crRNA targeting the Brucella bcsp31 gene.
- Developed fluorescence and lateral flow strip (LFS) assays based on the optimized RPA-CRISPR/Cas12a system.
- Evaluated assay performance including limit of detection (LoD), specificity, and concordance with qPCR using clinical samples.
Main Results:
- The RPA-CRISPR/Cas12a assay achieved an LoD of 1 copy/μL (fluorescence) and 10 copies/μL (LFS).
- The entire assay process was completed in under 30 minutes.
- Demonstrated excellent specificity against related pathogens and high concordance with qPCR in diverse clinical samples (blood, serum, milk, semen, vaginal secretions).
Conclusions:
- The developed RPA-CRISPR/Cas12a assay is a rapid, portable, reliable, and inexpensive tool for Brucella detection.
- This technology shows significant potential for field surveillance and clinical diagnostics of Brucellosis.
- Offers a promising alternative to conventional diagnostic methods, addressing current limitations in sensitivity, specificity, and speed.
