Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China.
Institute of Biological Engineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China.
Biotechnol Bioeng. 2021 Apr;118(4):1587-1596. doi: 10.1002/bit.27673. Epub 2021 Feb 4.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly throughout the whole world and caused significant difficulties in the prevention and control of the epidemic. In this case, several detection methods have been established based on nucleic acid diagnostic techniques and immunoassays to achieve sensitive and specific detection of SARS-CoV-2. However, most methods are still largely dependent on professional instruments, highly trained operators, and centralized laboratories. These limitations gravely diminish their practicality and portability. Herein, a clustered regularly interspaced short palindromic repeats (CRISPR) Cas12a based assay was developed for portable, rapid and sensitive of SARS-CoV-2. In this assay, samples were quickly pretreated and amplified by reverse transcription recombinase-aided amplification under mild conditions. Then, by combining the CRISPR Cas12a system and a glucose-producing reaction, the signal of the virus was converted to that of glucose, which can be quantitatively read by a personal glucose meter in a few seconds. Nucleocapsid protein gene was tested as a model target, and the sensitivity for quantitative detection was as low as 10 copies/μl, which basically meet the needs of clinical diagnosis. In addition, with the advantages of lower material cost, shorter detection time, and no requirement for professional instrument in comparison with quantitative reverse transcription-polymerase chain reaction, this assay is expected to provide a powerful technical support for the early diagnosis and intervention during epidemic prevention and control.
严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）已迅速在全球范围内传播，并对疫情的防控造成了重大困难。在这种情况下，已经基于核酸诊断技术和免疫测定法建立了几种检测方法，以实现对 SARS-CoV-2 的灵敏和特异性检测。然而，大多数方法仍然在很大程度上依赖于专业仪器、经过高度训练的操作人员和集中式实验室。这些局限性严重降低了它们的实用性和便携性。在此，开发了一种基于簇状规律间隔短回文重复序列（CRISPR）Cas12a 的测定法，用于 SARS-CoV-2 的便携式、快速和灵敏检测。在该测定法中，样品在温和条件下通过逆转录重组酶辅助扩增快速预处理和扩增。然后，通过结合 CRISPR Cas12a 系统和产糖反应，将病毒信号转换为葡萄糖信号，可在几秒钟内由个人血糖仪进行定量读取。核衣壳蛋白基因被用作模型靶标进行测试，定量检测的灵敏度低至 10 拷贝/μl，这基本满足了临床诊断的需求。此外，与定量逆转录-聚合酶链反应相比，该测定法具有较低的材料成本、更短的检测时间和不需要专业仪器的优势，有望为疫情防控期间的早期诊断和干预提供有力的技术支持。
