A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): Application for the diagnosis of COVID-19.
A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): Application for the diagnosis of COVID-19.
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DOI：
10.1016/j.talanta.2021.122591
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发表时间：
2021-10-01
期刊：
影响因子：
6.1
通讯作者：
Tai J
中科院分区：
文献类型：
--
作者：
Li S;Huang J;Ren L;Jiang W;Wang M;Zhuang L;Zheng Q;Yang R;Zeng Y;Luu LDW;Wang Y;Tai J
现有的CRISPR介导的诊断试验需要两个步骤(DNA或RNA扩增，然后CRISPR介导的序列特异性检测)来进行核酸检测，这增加了复杂性和样本交叉污染的风险。在这里，我们报告了一种新的CRISPR介导的测试，称为CRISPR-TOP(CRISPR-Mediated Testing in One-POT)，它将同时的靶标预扩增与CRISPR/Cas12b介导的检测集成到一锅反应混合物中，在恒温下进行。将新的CRISPR-TOP方法应用于严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)的检测，SARS-CoV-2是新冠肺炎(冠状病毒病2019)的病原体。新冠肺炎CRISPR-TOP针对SARS-CoV-2的ORF1ab(开放阅读框架1a/b)和NP(核蛋白)基因，在59℃下以最小仪器运行40分钟。新冠肺炎CRISPR-TOP分析可以在60分钟内返回结果，并且很容易通过视觉荧光或横向流动读数进行解释。新冠肺炎CRISPR-TOP的分析检测下限为每次反应10个拷贝(每个检测目标)，从非SARS-CoV-2模板中没有观察到交叉反应。在临床采集的非新冠肺炎标本中，该方法的特异性为100%(80/80例口咽拭子标本)。在收集的52份新冠肺炎阳性临床标本中，新冠肺炎CRISPR-TOP法荧光读数法38份阳性(73.1%)，侧流读数法35份(67.3%)阳性。这些诊断结果与RT-PCR的结果相似(34例阳性(65.4%))。这些数据表明，新冠肺炎CRISPR-TOP是一种简单、快速、准确和高灵敏度的检测SARS-CoV-2的方法，可用于资源短缺环境下的诊所、野外实验室和初级保健机构。
The existing CRISPR-mediated diagnostic tests require a two-step procedure (DNA or RNA amplification followed by CRISPR-mediated sequence-specific detection) for nucleic acid detection, which increases complexity and the risk of sample cross-contamination. Here, we report a new CRISPR-mediated test, called CRISPR-top (CRISPR-mediated testing in one-pot), which integrates simultaneous target pre-amplification with CRISPR/cas12b-mediated detection into a one-pot reaction mixture, performed at a constant temperature. The novel CRISPR-top assay was applied to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19 (coronavirus disease 2019). COVID-19 CRISPR-top targets the ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2, and operates at 59 °C for 40 min with minimal instrument. The COVID-19 CRISPR-top assay can return results within 60-min and is easily interpreted by visual fluorescence or lateral flow readouts. The analytical limit of detection (LoD) for COVID-19 CRISPR-top is 10 copies (for each detection target) per reaction with no cross-reactivity observed from non-SARS-CoV-2 templates. Among clinically collected non-COVID-19 samples, the assay's specificity was 100% (80/80 oropharynx swab samples). Among 52 COVID-19 positive clinical samples collected, the COVID-19 CRISPR-top assay yielded 38 (73.1%) positive results using fluorescence readout and 35 (67.3%) positive results with lateral-flow readout. These diagnostic results were similar to those obtained using RT-PCR (34 positive (65.4%)). These data indicate that COVID-19 CRISPR-top is a simple, rapid, accurate and highly sensitive method for SARS-CoV-2 detection which can be used in the clinic, field laboratories and primary care facilities in resource-challenged settings.
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DOI：
10.1126/science.aav4294
发表时间：
2018-11-16
期刊：
Science (New York, N.Y.)
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28.3
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