Shen Yu, Yi Chenchen, Wang Heng, Tang Yi, Li Jun
Hangzhou Center for Disease Control and Prevention (Hangzhou Health Supervision Institution), Hangzhou, China.
Zhejiang Key Laboratory of Multi-Omics in Infection and Immunity, Hangzhou, China.
Microbiol Spectr. 2025 Aug 27:e0099825. doi: 10.1128/spectrum.00998-25.
Brucellosis, a zoonotic disease caused by species, presents significant public health challenges due to its complex diagnosis and the limited availability of rapid detection methods. To address these challenges, we developed a novel detection method that integrates recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system, enabling dual readout through fluorescence (FL) and lateral flow strip (LFS) detection. The RPA-CRISPR/Cas12a-FL assay demonstrated an impressive detection limit of 1 copy/μL, which is 10 times more sensitive than quantitative polymerase chain reaction, while the RPA-CRISPR/Cas12a-LFS method achieved a detection limit of 10 copies/μL, comparable to nested PCR. Specificity testing confirmed the robustness of the assay, as it produced strong signals exclusively for without cross-reactivity with other bacterial species. Clinical validation using serum samples from 24 confirmed brucellosis patients and six healthy controls demonstrated a 100% concordance with serological results, underscoring the reliability of this method for clinical applications. This assay provides a rapid, sensitive, and specific tool for detection, suitable for both laboratory and field settings, and holds significant potential for enhancing the diagnosis and control of brucellosis.IMPORTANCEBrucellosis is a significant zoonotic disease, and rapid and accurate diagnosis is crucial for its treatment and control. To address this need, we developed a novel detection method that combines recombinant enzyme polymerase amplification with a CRISPR/Cas12a system, achieving dual readout through fluorescence and lateral flow strips. The test demonstrates excellent sensitivity and specificity, with clinical validation confirming complete concordance with serological results. This approach offers a fast, reliable, and field-deployable solution for brucellosis diagnosis, significantly enhancing disease management and public health outcomes.
布鲁氏菌病是一种由布鲁氏菌属物种引起的人畜共患病，因其诊断复杂且快速检测方法有限，给公共卫生带来了重大挑战。为应对这些挑战，我们开发了一种新型检测方法，该方法将重组酶聚合酶扩增（RPA）与CRISPR/Cas12a系统相结合，能够通过荧光（FL）和侧向流动试纸条（LFS）检测实现双重读数。RPA-CRISPR/Cas12a-FL检测方法的检测限低至1拷贝/μL，令人印象深刻，其灵敏度比定量聚合酶链反应高10倍，而RPA-CRISPR/Cas12a-LFS方法的检测限为10拷贝/μL，与巢式PCR相当。特异性测试证实了该检测方法的稳健性，因为它仅对布鲁氏菌产生强烈信号，与其他细菌物种无交叉反应。使用24例确诊布鲁氏菌病患者和6例健康对照的血清样本进行的临床验证表明，该方法与血清学结果的一致性达100%，突出了该方法在临床应用中的可靠性。该检测方法为布鲁氏菌检测提供了一种快速、灵敏且特异的工具，适用于实验室和现场环境，在加强布鲁氏菌病的诊断和控制方面具有巨大潜力。重要性布鲁氏菌病是一种重要的人畜共患病，快速准确的诊断对其治疗和控制至关重要。为满足这一需求，我们开发了一种新型检测方法，该方法将重组酶聚合酶扩增与CRISPR/Cas12a系统相结合，通过荧光和侧向流动试纸条实现双重读数。该测试显示出优异的灵敏度和特异性，临床验证证实与血清学结果完全一致。这种方法为布鲁氏菌病诊断提供了一种快速、可靠且可现场部署的解决方案，显著改善了疾病管理和公共卫生成果。
