普遍稳定且精确的 CRISPR-LAMP 检测平台，用于精确诊断多种呼吸道病毒，包括突变 SARS-CoV-2 Spike N501Y,Analytical Chemistry

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普遍稳定且精确的 CRISPR-LAMP 检测平台，用于精确诊断多种呼吸道病毒，包括突变 SARS-CoV-2 Spike N501Y
Analytical Chemistry ( IF 6.7 ) Pub Date : 2021-11-25 , DOI: 10.1021/acs.analchem.1c04065
Tong Zhang ₁ , Wei Zhao ₁ , Wang Zhao ₁ , Yuying Si ₂ , Nianzhen Chen ₂ , Xi Chen ₁ , Xinlian Zhang ₁ , Lieying Fan ₂ , Guodong Sui _{1,

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Affiliation

如今，快速准确地诊断呼吸道病毒是防止疫情再次爆发的迫切需要。为了克服这个问题，我们开发了一种成簇、规则间隔的短回文重复序列（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、甲型呼吸道合胞病毒、乙型呼吸道合胞病毒和重症急性呼吸道病毒。综合症冠状病毒2，包括关注的变种（B.1.1.7），它利用CRISPR相关蛋白12a（Cas12a）推进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，还开发了尿嘧啶-DNA-糖基化酶-逆转录-LAMP系统，该系统可以有效去除掺入dUTP的LAMP扩增子。体外Cas12a与28个crRNA的切割反应表明，LAMP扩增子中Cas12a/CRISPR RNA（crRNA）识别和切割没有位置限制，甚至LAMP扩增子的环状位置也能被有效识别和切割。野生型或尖峰 N501Y 的检测限为 10 个拷贝/μL（野生型），即使在背景（尖峰 N501Y）的比例水平为 1% 时也是如此。结合UDG-RT-LAMP技术、CRISPR-LAMP设计和突变检测设计，我们开发了CRISPR-LAMP检测平台，可以精确诊断病原体，稳定性更好，显着提高了点突变检测效率。

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Universally Stable and Precise CRISPR-LAMP Detection Platform for Precise Multiple Respiratory Tract Virus Diagnosis Including Mutant SARS-CoV-2 Spike N501Y

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.

更新日期：2021-12-07

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