Zhou Zhongqi, Lau Cia-Hin, Wang Jianchao, Guo Rui, Tong Sheng, Li Jiaqi, Dong Wenjiao, Huang Zhihao, Wang Tao, Huang Xiaojun, Yu Ziqing, Wei Chiju, Chen Gang, Xue Hongman, Zhu Haibao
Pediatric Hematology Laboratory, Division of Hematology/Oncology, Department of Pediatrics, The Seventh Affiliated Hospital of Sun Yat-Sen University, Shenzhen, Guangdong 518107, China.
Department of Biology, College of Science, Shantou University, Shantou, Guangdong 515063, China.
ACS Omega. 2024 Jun 21;9(26):28866-28878. doi: 10.1021/acsomega.4c03413. eCollection 2024 Jul 2.
To enable rapid and accurate point-of-care DNA detection, we have developed a single-step, amplification-free nucleic acid detection platform, a DNA substrate-mediated autocatalysis of CRISPR/Cas12a (DSAC). DSAC makes use of the trans-cleavage activity of Cas12a and target template-activated DNA substrate for dual signal amplifications. DSAC employs two distinct DNA substrate types: one that enhances signal amplification and the other that negatively modulates fluorescent signals. The positive inducer utilizes nicked- or loop-based DNA substrates to activate CRISPR/Cas12a, initiating trans-cleavage activity in a positive feedback loop, ultimately amplifying the fluorescent signals. The negative modulator, which involves competitor-based DNA substrates, competes with the probes for trans-cleaving, resulting in a signal decline in the presence of target DNA. These DNA substrate-based DSAC systems were adapted to fluorescence-based and paper-based lateral flow strip detection platforms. Our DSAC system accurately detected African swine fever virus (ASFV) in swine's blood samples at femtomolar sensitivity within 20 min. In contrast to the existing amplification-free CRISPR/Dx platforms, DSAC offers a cost-effective and straightforward detection method, requiring only the addition of a rationally designed DNA oligonucleotide. Notably, a common ASFV sequence-encoded DNA substrate can be directly applied to detect human nucleic acids through a dual crRNA targeting system. Consequently, our single-step DSAC system presents an alternative point-of-care diagnostic tool for the sensitive, accurate, and timely diagnosis of viral infections with potential applicability to human disease detection.
为实现快速、准确的即时DNA检测，我们开发了一种单步、无扩增的核酸检测平台——DNA底物介导的CRISPR/Cas12a自催化（DSAC）。DSAC利用Cas12a的反式切割活性和靶标模板激活的DNA底物进行双重信号放大。DSAC采用两种不同类型的DNA底物：一种增强信号放大，另一种对荧光信号进行负调控。正诱导剂利用带切口或成环的DNA底物激活CRISPR/Cas12a，在正反馈回路中启动反式切割活性，最终放大荧光信号。负调节剂涉及基于竞争物的DNA底物，与探针竞争反式切割，导致在靶标DNA存在时信号下降。这些基于DNA底物的DSAC系统适用于基于荧光和基于试纸条的侧向流动检测平台。我们的DSAC系统在20分钟内以飞摩尔灵敏度准确检测猪血液样本中的非洲猪瘟病毒（ASFV）。与现有的无扩增CRISPR/Dx平台相比，DSAC提供了一种经济高效且简单的检测方法，只需要添加合理设计的DNA寡核苷酸。值得注意的是，一个常见的ASFV序列编码的DNA底物可通过双crRNA靶向系统直接用于检测人类核酸。因此，我们的单步DSAC系统为敏感、准确和及时诊断病毒感染提供了一种替代的即时诊断工具，可能适用于人类疾病检测。
