Guan Xiaotian, Wang Peizheng, Wang Yi, Sun Shuqing
Institute of Biopharmaceutical and Healthcare Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China.
Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing 100010, China.
Biosensors (Basel). 2025 Jul 24;15(8):479. doi: 10.3390/bios15080479.
The CRISPR/Cas system has attracted increasing attention in accurate nucleic acid detection. Herein, we reported a CRISPR/Cas12a-chemiluminescence cascaded bioassay (CCCB) for the amplification-free and sensitive detection of human papillomavirus type 16 (HPV-16) and parvovirus B19 (PB-19). A magnetic bead (MB)-linking single-stranded DNA (LssDNA)-alkaline phosphatase (ALP) complex was constructed as the core component of the bioassay. During the detection process, the single-stranded target DNA was captured and enriched by LssDNA and then activated the trans-cleavage activity of Cas12a. Due to the Cas12a-mediated cleavage of LssDNA, ALP was released from the MB, subsequently catalyzing the substrate to generate a chemiluminescence (CL) signal. Given the cascade combination of CRISPR/Cas12a with the CL technique, the limits of detection for HPV-16 and PB-19 DNA were determined as 0.14 pM and 0.37 pM, respectively, and the whole detection could be completed within 60 min. The practicality and reliability of the platform were validated through target-spiked clinical specimens, and the recovery rate was 93.4-103.5%. This dual-amplification strategy-operating without target pre-amplification-featured high specificity, low contamination risk, facile preparation, and robust stability. It provides a novel approach for sensitive nucleic acid detection, with the potential for rapid extension to the diagnosis of various infectious diseases.
CRISPR/Cas系统在精确核酸检测中受到越来越多的关注。在此，我们报道了一种用于无扩增且灵敏检测人乳头瘤病毒16型（HPV - 16）和细小病毒B19（PB - 19）的CRISPR/Cas12a - 化学发光级联生物测定法（CCCB）。构建了一种磁珠（MB）连接单链DNA（LssDNA） - 碱性磷酸酶（ALP）复合物作为生物测定法的核心组件。在检测过程中，单链靶DNA被LssDNA捕获并富集，然后激活Cas12a的反式切割活性。由于Cas12a介导的LssDNA切割，ALP从磁珠上释放，随后催化底物产生化学发光（CL）信号。鉴于CRISPR/Cas12a与CL技术的级联组合，HPV - 16和PB - 19 DNA的检测限分别确定为0.14 pM和0.37 pM，整个检测可在60分钟内完成。通过添加靶标的临床标本验证了该平台的实用性和可靠性，回收率为93.4 - 103.5%。这种无需靶标预扩增的双扩增策略具有高特异性、低污染风险、制备简便和稳定性强的特点。它为灵敏核酸检测提供了一种新方法，具有快速扩展到各种传染病诊断的潜力。
