Zhongqi Zhou: Pediatric Hematology Laboratory, Division of Hematology/Oncology, Department of Pediatrics, The Seventh Affiliated Hospital of Sun Yat-Sen University, Shenzhen, Guangdong 518107, China.
Cia-Hin Lau: Department of Biology, College of Science, Shantou University, Shantou, Guangdong 515063, China. ORCID
Jianchao Wang: Department of Pathology, Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital, Fuzhou, Fujian 350014, China.
Rui Guo: Animal Husbandry and Veterinary Institute, Hubei Academy of Agricultural Science, Wuhan, Hubei 430064, China.
Sheng Tong: Department of Biomedical Engineering, University of Kentucky, Lexington, Kentucky 40506-0503, United States. ORCID
Jiaqi Li: Department of Biology, College of Science, Shantou University, Shantou, Guangdong 515063, China.
Wenjiao Dong: Department of Epidemiology and Health Statistics, School of Public Health, Guangdong Medical University, Dongguan, Guangdong 523808, China.
Zhihao Huang: Department of Biology, College of Science, Shantou University, Shantou, Guangdong 515063, China.
Tao Wang: Department of Biology, College of Science, Shantou University, Shantou, Guangdong 515063, China.
Ziqing Yu: Department of Pathology, Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital, Fuzhou, Fujian 350014, China.
Chiju Wei: Department of Biology, College of Science, Shantou University, Shantou, Guangdong 515063, China.
Gang Chen: Department of Pathology, Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital, Fuzhou, Fujian 350014, China.
Hongman Xue: Pediatric Hematology Laboratory, Division of Hematology/Oncology, Department of Pediatrics, The Seventh Affiliated Hospital of Sun Yat-Sen University, Shenzhen, Guangdong 518107, China.
Haibao Zhu: Department of Biology, College of Science, Shantou University, Shantou, Guangdong 515063, China.
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.
