Biology

CLC number: Q789

Detection of SARS-CoV-2 and Its Mutated Variants Using RT-LAMP-CRISPR-Cas12a Platform

基于CRISPR-Cas12a的环介导等温核酸扩增技术在SARS-CoV-2病毒检测中的应用

¹ Hubei Province Key Laboratory of Allergy and Immunology, Taikang Medical School (School of Basic Medical Sciences), Wuhan University, Wuhan 430071, Hubei, China
² Department of Laboratory Medicine, Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430016, Hubei, China
³ Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing 100029, China
⁴ Key Laboratory of Environmental Pollution Monitoring and Disease Control (Guizhou Medical University), Ministry of Education, Guiyang 550025, Guizhou, China

^† Corresponding author. E-mail: liuwanhong@whu.edu.cn (LIU W H); luxuan@whu.edu.cn (LU X)

Received: 14 April 2024

Abstract

The global outbreak of coronavirus disease 19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has raised significant global apprehension. Developing a rapid, efficient, sensitive, and accurate point-of-care detection method is imperative for curbing SARS-CoV-2 transmission. Here, we screened a sequence, designed a set of highly sensitive loop-mediated isothermal amplification primers (LAMP) and gRNA, and developed a user-friendly detection platform combining CRISPR-Cas12a and RT-LAMP technology to specifically detect SARS-CoV-2 and its 5 variants. Bioinformatics analysis and Cas12a-gRNA identification ensured sequence specificity, allowing us to identify SARS-CoV-2 mutations. We developed a method for the detection of SARS-CoV-2 using these primers in combination with LAMP amplification and CRISPR-Cas12a technology. This method is designed to detect SARS-CoV-2 (NC_045512), Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529). Additionally, it can differentiate SARS-CoV-2 from other coronaviruses. Quantitative analysis can be conducted by measuring fluorescence values, while qualitative analysis can be performed by observing fluorescence color point-of-care diagnosis changes with the naked eye. These results suggest that a set of novel sensitive LAMP primers and gRNA have been obtained to detect the extensive variants, and the RT-LAMP-CRISPR-Cas12a platform significantly facilitates point-of-care diagnosis, thereby halting the spread of SARS-CoV-2, thus contributing to COVID-19 prevention and control.

摘要

冠状病毒病由严重急性呼吸系统综合征冠状病毒2型(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)引起, 对全球公共卫生健康及经济发展造成严重威胁。发展一种快速、高效、敏感和准确的检测方法对遏制SARS-CoV-2的传播至关重要。本文结合环介导等温扩增(loop-mediated isothermal amplification primers, LAMP)和CRISPR-Cas12a技术, 经生物信息学分析筛选出SARS-CoV-2病毒不同株系的保守序列, 并设计一套高灵敏度高特异性的引物,开发了RT-LAMP-CRISPR-Cas12a检测平台,其可特异性检测SARS-CoV-2及其变异株。该平台可检测SARS-CoV-2 (NC_045512)、Alpha (B.1.1.7)、Beta (B.1.351)、Gamma (P.1)、Delta (B.1.617.2)和Omicron (B.1.1.529)等突变株, 通过测量荧光值进行定量分析。这些结果为该类冠状病毒再次爆发时的预防和治疗提供了一种新的检测技术平台和实验数据。