RT-RAA联合CRISPR/Cas12a快速检测新型冠状病毒方法的建立与评价
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惠州市科技研发计划社会发展领域研发项目( 210426104574869 )


Establishment and evaluation of RT-RAA combined with CRISPR/Cas12a for rapid detection of SA RS-CoV-2
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    摘要:

    摘要:目的建立一种基于反转录酶一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10°~ 10 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/Cas1 2a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 C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1x 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/Cas 12a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。

    Abstract:

    Abstract: 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)/Cas 12a 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 LbCas 12a reaction temperature. The sensitivity and specificity of the method were evaluated using recombinant plasmids ( 10*-10* copies/ μL) and other respiratory pathogens. The RT-RAA-CRISPR/Cas 1 2a method was compared with RT-PCR by testing 70 dlinical samples in parallel. Results The optimized RT-RAA-CRISPR/ Cas 12a assay could detect SARS-CoV-2 within 50 min at 37 9C. The limit of detection was 10 copies/ μL for the fluorescence -based method and 1x 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 performned 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.

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章太婵,车玉传,梁雪雁,韦华贵,范向平,黄承仕,林敏,陈江涛. RT-RAA联合CRISPR/Cas12a快速检测新型冠状病毒方法的建立与评价[J].临床检验杂志,2024,42(4):246-251

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  • 收稿日期:2023-07-09
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  • 在线发布日期: 2024-06-14
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