Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19
Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19
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DOI：
10.1371/journal.ppat.1008705
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发表时间：
2020-08-01
期刊：
影响因子：
6.7
通讯作者：
Xu, Teng
中科院分区：
文献类型：
--
作者：
Hou, Tieying;Zeng, Weiqi;Xu, Teng
SARS冠状病毒2是第三种人畜共患冠状病毒，近年来在全球范围内引起了人们的极大关注。这种新型病原体的快速准确诊断不仅在临床上而且在技术上都面临巨大挑战。宏基因组下一代测序（mNGS）和逆转录PCR（RT-PCR）是最常用的分子生物学方法。然而，每种方法都有其自身的局限性。在这项研究中，我们开发了一种等温的、基于CRISPR的COVID-19诊断方法，具有接近单拷贝的灵敏度。还比较了所有三种技术平台的诊断性能。我们的研究旨在为SARS-CoV-2的分子检测提供更多见解，并为这种新出现的病毒提供新的诊断选择。作者摘要最近发现的β冠状病毒严重急性呼吸道综合征（SARS）-CoV-2引起了2019冠状病毒病（COVID-19）的爆发，并迅速发展成为全球大流行。对快速筛查和识别COVID-19的需求激增，带来了巨大的诊断挑战。缺乏快速和准确的分子工具阻碍了对病毒威胁的有效公共卫生反应。在这里，我们利用可编程CRISPR/Cas 13 a的独特附带活性来开发CRISPR-COVID，这是一种快速灵敏的SARS-CoV-2感染诊断方法，并将其与临床队列中基于测序的宏基因组和基于RT-PCR的检测方法进行了比较。CRISPR-COVID表现出接近单拷贝的敏感性水平，并且具有高度特异性，不会与相关病原体发生交叉反应。CRISPR-COVID仅需40分钟，不需要复杂的热循环仪，为传统的RT-PCR检测提供了一种有价值的替代方案，以规避COVID-19检测的检测周转时间、设备和试剂供应方面的瓶颈。
The recent outbreak of human infections caused by SARS-CoV-2, the third zoonotic coronavirus has raised great public health concern globally. Rapid and accurate diagnosis of this novel pathogen posts great challenges not only clinically but also technologically. Metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methodologies. However, each has their own limitations. In this study, we developed an isothermal, CRISPR-based diagnostic for COVID-19 with near single-copy sensitivity. The diagnostic performances of all three technology platforms were also compared. Our study aimed to provide more insights into the molecular detection of SARS-CoV-2, and also to present a novel diagnostic option for this new emerging virus.Author summary The recently discovered betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 has caused an outbreak of Coronavirus Disease 2019 (COVID-19) that rapidly developed into a global pandemic. The surging demand for rapid screening and identification of COVID-19 posts great diagnostic challenges. A lack of rapid and accurate molecular tools has hampered efficient public health responses to the viral threat. Here, we harnessed the unique collateral activity of programmable CRISPR/Cas13a to develop CRISPR-COVID, a rapid and sensitive diagnostic for SARS-CoV-2 infection, and compared it to sequencing-based metagenomic and RT-PCR-based assays in a clinical cohort. CRISPR-COVID demonstrated a sensitivity level of near single copy and was highly specific without cross reacting to related pathogens. CRISPR-COVID takes only 40 mins and requires no sophiscated thermo-cyclers, providing a valuable alternative to the conventional RT-PCR assay to circumvent the bottlenecks in assay turnaround time, equipment and reagent supplies for COVID-19 testing.