Establishment and evaluation of RT-RAA combined with CRISPR/Cas12a for rapid detection of SARS-CoV-2
10.13602/j.cnki.jcls.2024.04.02
- VernacularTitle:RT-RAA联合CRISPR/Cas12a快速检测新型冠状病毒方法的建立与评价
- Author:
Taichan ZHANG
1
;
Yuchuan CHE
;
Xueyan LIANG
;
Huagui WEI
;
Xiangping FAN
;
Chengshi HUANG
;
Min LIN
;
Jiangtao CHEN
Author Information
1. 惠州市中心人民医院检验科,广东惠州 516008
- Keywords:
SARS-CoV-2;
reverse transcriptase-recombinase aided amplification;
CRISPR/Cas12a;
lateral flow assay;
rapid detection
- From:
Chinese Journal of Clinical Laboratory Science
2024;42(4):246-251
- CountryChina
- Language:Chinese
-
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)/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 tempera-ture and time and LbCas12a reaction temperature.The sensitivity and specificity of the method were evaluated using recombinant plas-mids(100-106 copies/μL)and other respiratory pathogens.The RT-RAA-CRISPR/Cas12a method was compared with RT-PCR by tes-ting 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.
