数字农科院2.0

Rapid and Specific Detection of Active SARS-CoV-2 With CRISPR/Cas12a

文献类型： 外文期刊

作者： Xinyi Liu;Yanhua Li;Xin Wang;Yifan Song;Lina Wu;Benyuan Yu;Xiaodong Ma;Peixiang Ma;Ming Liu;Xingxu Huang;Xinjie Wang

作者机构：

关键词： COVID-19;CRISPR-based detection;CRISPR/Cas12a;SARS-CoV-2;virus subgenome

期刊名称： Frontiers in Microbiology

ISSN： 1664-302X

年卷期： 2022 年 12 卷

页码：

收录情况： JCR（2021版）

摘要： Rapid and sensitive nucleic acid detection of SARS-CoV-2 has contributed to the clinical diagnosis and control of COVID-19. Although detection of virus genomic RNA (gRNA) has been commonly used in clinical diagnosis, SARS-CoV-2 gRNA detection could not discriminate between active infectious virus with remnant viral RNA. In contrast to genomic RNA, subgenomic RNAs (sgRNAs) are only produced when the virus is actively replicating and transcription, detection of sgRNA could be an indication to evaluate infectivity. CRISPR/Cas-based nucleic acid detection methods have been considered potential diagnostic tools due to their intrinsic sensitivity, specificity and simplicity. In this study, to specifically detect active virus replication, we developed a CRISPR-based active SARS-CoV-2 (CRISPR-actCoV) detection strategy by detecting sgRNAs of SARS-CoV-2. CRISPR-actCoV with CRISPR Cas12a-assisted fluorescence reporter system enables detection of sgRNAs at 10 copies in 35 min with high specificity and can be read out with naked eyes. Further, we performed CRISPR-actCoV mediated sgRNA detection in 30 SARS-CoV-2 potentially infected clinical samples, and 21 samples were SARS-CoV-2 sgRNA positive. A quantitative RT-PCR assay was also performed to detect gRNA of SARS-CoV-2 in parallel. Among the 30 clinical samples, 27 samples were gRNA positive. Taken together, CRISPR-actCoV provides an alternative for rapid and accurate detection of active SARS-CoV-2 and has great significance in better response of coronavirus causing epidemic disease.

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