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 act ive 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.
【저자키워드】 COVID-19, SARS-CoV-2, CRISPR/Cas12a, CRISPR-based detection, virus subgenome, 【초록키워드】 coronavirus, Transcription, diagnostic, virus, CRISPR, clinical samples, sensitivity, specificity, Epidemic, Nucleic acid detection, subgenomic RNA, Rapid, sgRNA, virus replication, Viral RNA, reporter, disease, Clinical diagnosis, Quantitative, RT-PCR assay, Infectious virus, genomic RNA, sgRNAs, eyes, positive, gRNA, SARS- CoV, intrinsic, produced, performed, detect, evaluate, provide, contributed, 【제목키워드】 detection, Rapid, Specific,