Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3), Department of Environmental Science and Engineering, Fudan University, 2205 Songhu Road, Shanghai 200433, P. R. China.
Department of Clinical Laboratory, Shanghai East Hospital, School of Medicine, Tong Ji University, 150 Ji Mo Road, Shanghai 200120, P. R. China.
Anal Chem. 2021 Dec 7;93(48):16184-16193. doi: 10.1021/acs.analchem.1c04065. Epub 2021 Nov 25.
Nowadays, rapid and accurate diagnosis of respiratory tract viruses is an urgent need to prevent another epidemic outbreak. To overcome this problem, we have developed a clustered, regularly interspaced short palindromic repeats (CRISPR) loop mediated amplification (LAMP) technology to detect influenza A virus, influenza B virus, respiratory syncytial A virus, respiratory syncytial B virus, and severe acute respiratory syndrome coronavirus 2, including variants of concern (B.1.1.7), which utilized CRISPR-associated protein 12a (Cas12a) to advance LAMP technology with the sensitivity increased 10 times. To reduce aerosol contamination in CRISPR-LAMP technology, an uracil-DNA-glycosylase-reverse transcription-LAMP system was also developed which can effectively remove dUTP-incorporated LAMP amplicons. In vitro Cas12a cleavage reaction with 28 crRNAs showed that there were no position constraints for Cas12a/CRISPR RNA (crRNA) recognition and cleavage in LAMP amplicons, and even the looped position of LAMP amplicons could be effectively recognized and cleaved. Wild-type or spike N501Y can be detected with a limit of detection of 10 copies/μL (wild-type) even at a 1% ratio level on the background (spike N501Y). Combining UDG-RT-LAMP technology, CRISPR-LAMP design, and mutation detection design, we developed a CRISPR-LAMP detection platform that can precisely diagnose pathogens with better stability and significantly improved point mutation detection efficiency.
现如今，快速准确地诊断呼吸道病毒是防止再次爆发疫情的当务之急。为了解决这个问题，我们开发了一种成簇、规律间隔短回文重复序列（CRISPR）环介导扩增（LAMP）技术，用于检测甲型流感病毒、乙型流感病毒、呼吸道合胞病毒 A 型、呼吸道合胞病毒 B 型和严重急性呼吸综合征冠状病毒 2，包括关切变异株（B.1.1.7），该技术利用 CRISPR 相关蛋白 12a（Cas12a）将 LAMP 技术灵敏度提高了 10 倍。为了减少 CRISPR-LAMP 技术中的气溶胶污染，还开发了一种尿嘧啶-DNA-糖基化酶-逆转录-LAMP 系统，可有效去除含 dUTP 的 LAMP 扩增产物。体外 Cas12a 与 28 个 crRNA 的切割反应表明，Cas12a/CRISPR RNA（crRNA）识别和切割 LAMP 扩增产物没有位置限制，甚至可以有效识别和切割 LAMP 扩增产物的环化位置。即使在背景（刺突 N501Y）中 1%的比例水平下，也可以检测到 10 拷贝/μL（野生型）的野生型或刺突 N501Y。通过将 UDG-RT-LAMP 技术、CRISPR-LAMP 设计和突变检测设计相结合，我们开发了一种 CRISPR-LAMP 检测平台，该平台可以更稳定地精确诊断病原体，并显著提高点突变检测效率。
