Establishment of a rapid Brucella detection method based on MCDA-CRISPR dual signal amplification system for reducing transfusion-transmitted diseases

Abstract Brucellosis is a common zoonotic disease caused by Brucella and remains a globally concerning public health issue. Timely and effective detection methods are crucial for clinical diagnosis. We developed a novel Brucella detection platform (MCDA-CRISPR) by integrating multiple cross displacement amplification (MCDA) with a CRISPR-Cas12a-based biosensing system, and preliminarily applied it for the first time to screen for Brucella in voluntary blood donors from Xinjiang, China. This technology enables amplification under isothermal conditions at 64 °C using only a water bath, requires no specialized equipment, and completes detection within 60 min. Amplification products can be directly visualized under UV light without complex interpretation. Performance results demonstrated a minimum detection limit of 1 fg/μL for Brucella DNA, making the method 100 times more sensitive than conventional PCR. The assay showed 100% specificity for Brucella detection with no cross-reactivity to non-Brucella pathogens. The assay could also detect Brucella in blood donors samples and showed the same sensitivity and specificity as the culture method. The assay is a visual, sensitive, and highly specific detection technique. When applied to routine blood transfusion screening in areas with high prevalence of brucellosis, such as Xinjiang, can effectively reduce the risk of transfusion-transmitted brucellosis, and hold broad application prospects in resource-limited primary or field testing scenarios.