RT-RAA联合CRISPR/Cas12a快速检测新型冠状病毒方法的建立与评价  

Establishment and evaluation of RT-RAA combined with CRISPR/Cas12a for rapid detection of SARS-CoV-2

作　　者：章太婵 车玉传 梁雪雁 韦华贵 范向平[1] 黄承仕 林敏[2,3] 陈江涛 ZHANG Taichan;CHE Yuchuan;LIANG Xueyan;WEI Huagui;FAN Xiangping;HUANG Chengshi;LIN Min;CHEN Jiangtao(Department of Clinical Laboratory,Huizhou Central People's Hospital,Huizhou 516008,Guangdong;Department of Clinical Laboratory,Chaozhou People's Hospital Affiliated to Shantou University,Chaozhou 521011,Guangdong;Department of Clinical Laboratory,the Affiliated Hospital of Youjiang Medical University for Nationalities,Baise 533099,Guangxi,China)

机构地区：[1]惠州市中心人民医院检验科,广东惠州516008 [2]汕头大学附属潮州市人民医院检验科,广东潮州521011 [3]右江民族医学院附属医院检验科,广西百色533099

出　　处：《临床检验杂志》2024年第4期246-251,共6页Chinese Journal of Clinical Laboratory Science

基　　金：惠州市科技研发计划社会发展领域研发项目(210426104574869)。

摘　　要：目的建立一种基于反转录酶-重组酶介导等温核酸扩增技术(RT-RAA)联合簇状规则间隔的短回文重复序列(CRISPR)/Cas12a系统的快速检测新型冠状病毒(SARS-CoV-2)的方法,并对其进行评价。方法根据NCBI数据库中SARS-CoV-2的核衣壳(N)基因设计RT-RAA引物和CRISPR来源RNA(crRNA),优化检测体系中乙酸镁(MgAc)用量、RT-RAA反应温度和时间以及LbCas12a反应温度。以100~106 copies/μL重组质粒和其他呼吸道病原体分别评估该方法灵敏度和特异性。采用RT-RAA-CRISPR/Cas12a法和RT-PCR法对70份临床标本进行平行检测,比较两种方法的符合率。结果优化后的RT-RAA-CRISPR/Cas12a检测方法可在37℃条件下50 min内完成SARS-CoV-2检测。荧光法检测灵敏度为10 copies/μL,侧流层析法为1×102 copies/μL。该方法能特异检测SARS-CoV-2,与其他呼吸道病原体无交叉反应。RT-RAA-CRISPR/Cas12a法检测70份临床标本的结果与RT-PCR法完全一致,符合率为100%。结论建立的RT-RAA-CRISPR/Cas12a法检测SARS-CoV-2快速、经济、灵敏度高、特异性强,无需昂贵仪器设备,可由经验不足的人员操作,为临床快速诊断和现场大规模筛查SARS-CoV-2提供了新的思路和方法。Objective To establish and evaluate a rapid detection method for SARS-CoV-2 based on reverse transcriptase-recombinase aided amplification(RT-RAA)combined with the clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a system.Methods RT-RAA primers and CRISPR-derived RNA(crRNA)were designed based on the nucleocapsid(N)gene of SARS-CoV-2 from NCBI database.The detection system was optimized with magnesium acetate(MgAc)concentration,RT-RAA reaction temperature and time and LbCas12a reaction temperature.The sensitivity and specificity of the method were evaluated using recombinant plasmids(100-106 copies/μL)and other respiratory pathogens.The RT-RAA-CRISPR/Cas12a method was compared with RT-PCR by testing 70 clinical samples in parallel.Results The optimized RT-RAA-CRISPR/Cas12a assay could detect SARS-CoV-2 within 50 min at 37℃.The limit of detection was 10 copies/μL for the fluorescence-based method and 1×102 copies/μL for the lateral flow assay.The method specifically detected SARS-CoV-2 without cross-reactivity to other respiratory pathogens.The results of testing 70 clinical samples using RT-RAA-CRISPR/Cas12a showed agreement of 100%with those of RT-PCR.Conclusion The established RT-RAA-CRISPR/Cas12a assay for SARS-CoV-2 detection is rapid,cost-effective,highly sensitive and specific.It can be performed by less experienced personnel and no expensive equipment is required,thus it may provide a new approach for rapid clinical diagnosis and large-scale on-site screening of SARS-CoV-2.

关 键 词：新型冠状病毒 重组酶介导等温扩增 CRISPR/Cas12a 侧流层析 快速检测 

分 类 号：R446[医药卫生—诊断学] R373.1[医药卫生—临床医学]