环介导等温扩增
放大器
严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)
分子诊断学
2019年冠状病毒病(COVID-19)
病毒学
检出限
冠状病毒
肉眼
计算生物学
化学
生物
DNA
聚合酶链反应
传染病(医学专业)
医学
基因
生物信息学
疾病
色谱法
遗传学
病理
作者
Rui Wang,Chunyan Qian,Yanan Pang,Miaomiao Li,Yu Yang,Haijing Ma,Manying Zhao,Feng Qian,Hang Yu,Zhenping Liu,Ting Ni,Zheng Yan,Yongming Wang
标识
DOI:10.1016/j.bios.2020.112766
摘要
The 2019 novel coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected all aspects of human life. Rapid, accurate, sensitive and user friendly detection method is urgently needed to facilitate early intervention and control the spread of SARS-CoV-2. Here, we propose a one-pot visual SARS-CoV-2 detection system named "opvCRISPR" by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12a cleavage in a single reaction system. We demonstrate that the collateral activity against single-stranded DNA (ssDNA) reporters of activated Cas12a triggered by RT-LAMP amplicon increases detection sensitivity and makes detection results observable with naked eye. The opvCRISPR enables detection at nearly single molecule level in 45 min. We validate this method with 50 SARS-CoV-2 potentially infected clinical samples. The opvCRISPR diagnostic results provide 100% agreement with the Centers for Disease Control and Prevention (CDC)-approved quantitative RT-PCR assay. The opvCRISPR holds great potential for SARS-CoV-2 detection in next-generation point-of-care molecular diagnostics.
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