期刊
BIOSENSORS & BIOELECTRONICS
卷 169, 期 -, 页码 -出版社
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2020.112642
关键词
SARS-CoV-2; RT-LAMP; CRISPR/Cas12a; Visual detection
类别
资金
- National Natural Science Foundation of China [31571918]
- Science and Technology Department of Zhejiang Province, China [2020C03123, LGN20C050001]
- Key Program of Logistics Research of China [BWS17J030]
- National Science and Technology Major Project for Major New Drugs Innovation and Development [2015zx09j15102-002]
The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method for SARS-CoV-2 with LAMP and CRISPR/Cas12a technology. CRISPR/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR/Casl 2a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5'-6FAM 3'-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method.
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