Yuan Ting, Yuan Jianbo, Huang Jian, Li Nana, Cai Huan, Yang Yan, Li Juan, Chen Rui, Min Xun
Department of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi, China.
School of Laboratory Medicine, Zunyi Medical University, Zunyi, China.
Front Chem. 2025 Aug 7;13:1631086. doi: 10.3389/fchem.2025.1631086. eCollection 2025.
The rapid and accurate identification of (MTB) is essential for effective tuberculosis (TB) control. However, conventional diagnostic methods for MTB suffer from limitations such as low sensitivity, poor specificity, high cost, reliance on specialized instruments, and complex, time-consuming procedures. To address these challenges, there is an urgent need for a simple, rapid, and highly sensitive detection method that can be deployed in point-of-care settings.
We developed a one-pot biosensing system combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a-driven G4-hemin self-assembly for the colorimetric detection of MTB. Glycerol was employed as a phase-separation barrier to prevent interference between RPA amplification and CRISPR/Cas12a trans-cleavage. A single-stranded DNA (ssDNA) probe, designed to self-assemble with ssDNA-hemin into G4-hemin nanozymes upon CRISPR/Cas12a-mediated cleavage, served as the reaction substrate. The ssDNA-hemin further enhanced the catalytic activity of the generated G4-hemin DNAzyme. The entire assay was completed in a single step within 60 min without requiring complex instrumentation.
Under optimized conditions, the biosensing system achieved ultrasensitive naked-eye detection of MTB with a limit of detection (LOD) of 10 copies/μL, comparable to traditional four-step fluorescent assays. Clinical validation using 104 patient samples demonstrated high concordance with standard diagnostic methods. This approach combines the advantages of recombinase polymerase amplification (RPA), CRISPR/Cas12a specificity, and G4-hemin DNAzyme-based colorimetric signal amplification, enabling simple, equipment-free visual detection. Given its speed, sensitivity, and ease of use, this biosensing system holds significant promise for point-of-care MTB nucleic acid testing in resource-limited settings.
快速准确地鉴定结核分枝杆菌（MTB）对于有效控制结核病（TB）至关重要。然而，MTB的传统诊断方法存在局限性，如灵敏度低、特异性差、成本高、依赖专业仪器以及操作复杂、耗时等。为应对这些挑战，迫切需要一种可在即时检测环境中部署的简单、快速且高度灵敏的检测方法。
我们开发了一种一锅式生物传感系统，该系统将重组酶聚合酶扩增（RPA）与CRISPR/Cas12a驱动的G4-血红素自组装相结合，用于MTB的比色检测。甘油用作相分离屏障，以防止RPA扩增与CRISPR/Cas12a反式切割之间的干扰。一种单链DNA（ssDNA）探针，经设计可在CRISPR/Cas12a介导的切割后与ssDNA-血红素自组装形成G4-血红素纳米酶，用作反应底物。ssDNA-血红素进一步增强了所生成的G4-血红素脱氧核酶的催化活性。整个检测在60分钟内一步完成，无需复杂仪器。
在优化条件下，该生物传感系统实现了对MTB的超灵敏肉眼检测，检测限（LOD）为10拷贝/μL，与传统的四步荧光检测相当。使用104份患者样本进行的临床验证表明，该方法与标准诊断方法高度一致。这种方法结合了重组酶聚合酶扩增（RPA）的优势、CRISPR/Cas12a的特异性以及基于G4-血红素脱氧核酶的比色信号放大，实现了无需设备的简单视觉检测。鉴于其速度、灵敏度和易用性，这种生物传感系统在资源有限的环境中进行即时MTB核酸检测具有巨大潜力。
