Digital CRISPR/Cas-Assisted Assay for Rapid and Sensitive Detection of SARS-CoV-2.
Digital CRISPR/Cas-Assisted Assay for Rapid and Sensitive Detection of SARS-CoV-2.
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DOI：
10.1002/advs.202003564
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发表时间：
2021-03
期刊：
影响因子：
--
通讯作者：
Wang TH
中科院分区：
文献类型：
--
作者：
Park JS;Hsieh K;Chen L;Kaushik A;Trick AY;Wang TH
为应对COVID-19大流行，对快速诊断的前所未有的需求使人们关注成簇规则间隔短回文重复序列（CRISPR）/CRISPR相关系统（Cas）辅助的核酸检测分析。这些分析已经受益于优雅的检测机制、快速的分析时间和较低的反应温度，可以通过与强大的数字化检测集成来进一步推进。因此，第一个数字化CRISPR/Cas辅助分析数字化-增强型CRISPR/Cas辅助一锅病毒检测（deCOViD）-被开发并应用于SARS-CoV-2检测。deCOViD是通过将一步式、基于荧光的CRISPR/Cas 12 a辅助逆转录重组酶聚合酶扩增测定法调整和离散化到市售微流体数字芯片内的亚纳升反应威尔斯孔中来实现的。均匀升高的数字浓度使deCOViD能够在<15分钟内实现定性检测，并在30分钟内实现定量检测，具有高信号背景比，宽动态范围和高灵敏度-低至1基因组当量（GE）µL−1的SARS-CoV-2 RNA和20 GE µL−1的热灭活SARS-CoV-2，它超越了基于台式的同类产品，代表了迄今为止最快，最灵敏的CRISPR/Cas辅助SARS-CoV-2检测之一。此外，deCOViD可以检测临床样本中的RNA提取物。综上所述，deCOViD为推进CRISPR/Cas辅助检测和抗击COVID-19大流行及其他疾病开辟了一条新途径。通过在商业微流体数字芯片的亚纳升反应威尔斯孔内离散一步、基于荧光的CRISPR/Cas 12 a辅助的逆转录重组酶聚合酶扩增测定来创建数字成簇规则间隔短回文重复序列（CRISPR）/Cas 12 a辅助的核酸检测测定，它可以在30分钟内定量检测1个基因组当量µL−1的SARS‐CoV‐2 RNA-这是迄今为止最快和最灵敏的CRISPR/Cas辅助检测之一。
The unprecedented demand for rapid diagnostics in response to the COVID‐19 pandemic has brought the spotlight onto clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated systems (Cas)‐assisted nucleic acid detection assays. Already benefitting from an elegant detection mechanism, fast assay time, and low reaction temperature, these assays can be further advanced via integration with powerful, digital‐based detection. Thus motivated, the first digital CRISPR/Cas‐assisted assay—coined digitization‐enhanced CRISPR/Cas‐assisted one‐pot virus detection (deCOViD)—is developed and applied toward SARS‐CoV‐2 detection. deCOViD is realized through tuning and discretizing a one‐step, fluorescence‐based, CRISPR/Cas12a‐assisted reverse transcription recombinase polymerase amplification assay into sub‐nanoliter reaction wells within commercially available microfluidic digital chips. The uniformly elevated digital concentrations enable deCOViD to achieve qualitative detection in <15 min and quantitative detection in 30 min with high signal‐to‐background ratio, broad dynamic range, and high sensitivity—down to 1 genome equivalent (GE) µL−1 of SARS‐CoV‐2 RNA and 20 GE µL−1 of heat‐inactivated SARS‐CoV‐2, which outstrips its benchtop‐based counterpart and represents one of the fastest and most sensitive CRISPR/Cas‐assisted SARS‐CoV‐2 detection to date. Moreover, deCOViD can detect RNA extracts from clinical samples. Taken together, deCOViD opens a new avenue for advancing CRISPR/Cas‐assisted assays and combating the COVID‐19 pandemic and beyond. A digital clustered regularly interspaced short palindromic repeats (CRISPR)/Cas‐assisted nucleic acid detection assay is created by discretizing a one‐step, fluorescence‐based, CRISPR/Cas12a‐assisted reverse transcription recombinase polymerase amplification assay within sub‐nanoliter reaction wells of a commercial microfluidic digital chip, which enables quantitative detection of 1 genome equivalent µL−1 of SARS‐CoV‐2 RNA in <30 min—one of the fastest and most sensitive CRISPR/Cas‐assisted detection to date.
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