Rapid and Amplification-free Nucleic Acid Detection with DNA Substrate-Mediated Autocatalysis of CRISPR/Cas12a
文献类型： 外文期刊
作者： Zhou, Zhongqi 1 ; Lau, Cia-Hin 2 ; Wang, Jianchao 3 ; Guo, Rui 4 ; Tong, Sheng 6 ; Li, Jiaqi 2 ; Dong, Wenjiao 7 ; Huang, Zhihao 2 ; Wang, Tao 2 ; Huang, Xiaojun 8 ; Yu, Ziqing 3 ; Wei, Chiju 2 ; Chen, Gang 3 ; Xue, Hongman 1 ; Zhu, Haibao 2 ;
作者机构： 1.Sun Yat Sen Univ, Affiliated Hosp 7, Dept Pediat, Div Hematol Oncol,Pediat Hematol Lab, Shenzhen 518107, Guangdong, Peoples R China
2.Shantou Univ, Coll Sci, Dept Biol, Shantou 515063, Guangdong, Peoples R China
3.Fujian Med Univ, Clin Oncol Sch, Dept Pathol, Fuzhou 350014, Fujian, Peoples R China
4.Hubei Acad Agr Sci, Anim Husb & Vet Inst, Wuhan 430064, Hubei, Peoples R China
5.Minist Agr, Key Lab Prevent & Control Agents Anim Bacteriosis, Wuhan 430064, Hubei, Peoples R China
6.Univ Kentucky, Dept Biomed Engn, Lexington, KY 40506 USA
7.Guangdong Med Univ, Sch Publ Hlth, Dept Epidemiol & Hlth Stat, Dongguan 523808, Guangdong, Peoples R China
8.Xiamen Fly Gene Biomed Technol CO LTD, Xiamen 361000, Fujian, Peoples R China
期刊名称：ACS OMEGA （ 影响因子：3.7； 五年影响因子：4.0 ）
ISSN： 2470-1343
年卷期： 2024 年 9 卷 26 期
页码：
收录情况： SCI
摘要： 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.
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