A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection.
A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection.
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DOI：
10.1016/j.ebiom.2020.103036
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发表时间：
2020-11
期刊：
影响因子：
11.1
通讯作者：
Wang J
中科院分区：
文献类型：
--
作者：
Huang W;Yu L;Wen D;Wei D;Sun Y;Zhao H;Ye Y;Chen W;Zhu Y;Wang L;Wang L;Wu W;Zhao Q;Xu Y;Gu D;Nie G;Zhu D;Guo Z;Ma X;Niu L;Huang Y;Liu Y;Peng B;Zhang R;Zhang X;Li D;Liu Y;Yang G;Liu L;Zhou Y;Wang Y;Hou T;Gao Q;Li W;Chen S;Hu X;Han M;Zheng H;Weng J;Cai Z;Zhang X;Song F;Zhao G;Wang J
自新冠肺炎大流行开始以来，实时逆转录聚合酶链式反应一直是最有效和被广泛应用的诊断技术。然而，经常会遇到Ct值较高的模糊RRT-PCR读数，导致诊断的不确定性。根据SARS-CoV-2Orf1ab(O)和N片段的RRT-PCR扩增产物特异性地触发Cas12a的反式切割活性，开发了一种特异性的聚合酶链式反应扩增核酸增强子。SENA首先用纯SARS-CoV-2 RNA标准进行系统的滴定实验，以确定其敏感性和特异性，然后在几家医院进行验证，使用几种商业RRT-PCR试剂盒，并在不同情况下测试各种临床标本。为了定量分析靶基因O和N，定义了荧光变化(FC)与混合SENA反应的比值(MIX-FC Ratio)，95%可信区间的MIX-FC Ratio的检测下限为1.2≤1.6≤2.1。共分析了295份临床标本，其中21份不确定的RRT-PCR标本以及4份假阴性和2份假阳性标本被SENA鉴定，并经下一代测序(NGS)进一步验证。MIX-FC比值的分界值为1.145为阳性，1.068为阴性。SENA同时提高了逆转录聚合酶链式反应的敏感性和特异性，解决了新冠肺炎诊断中的不确定性问题，从而为抗击疫情提供了一种简单、低成本的辅助诊断方法。详细的资金信息可在手稿的末尾找到。
Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. Detailed funding information is available at the end of the manuscript.
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DOI：
10.1126/science.aav4294
发表时间：
2018-11-16
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Harrington LB;Burstein D;Chen JS;Paez-Espino D;Ma E;Witte IP;Cofsky JC;Kyrpides NC;Banfield JF;Doudna JA
通讯作者：
Doudna JA
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
CRISPR-Cas12a辅助核酸检测
影响因子：
33.5
作者：
Li SY;Cheng QX;Wang JM;Li XY;Zhang ZL;Gao S;Cao RB;Zhao GP;Wang J
通讯作者：
Wang J
影响因子：
4.7
作者：
Li, Linxian;Li, Shiyuan;Wang, Jin
通讯作者：
Wang, Jin
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle