Visually amplification-free rapid detection of 2019-nCoV nucleic acid based on CRISPR/Cas13a
10.3760/cma.j.cn114452-20231225-00375
- VernacularTitle:基于CRISPR/Cas13a的新型冠状病毒核酸免扩增可视化快速检测研究
- Author:
Nan ZHAO
1
;
Yong QI
;
Wei LI
;
Yingqing MAO
;
Wenjing LIU
;
Yifang HAN
;
Erxin ZHANG
;
Yingjia XU
;
Ruichen LYU
;
Yuxin JIANG
;
Yuzhen LAI
;
Jiameng LI
;
Wanpeng SHEN
;
Yue SONG
;
Yuexi LI
Author Information
1. 南京医科大学公共卫生学院流行病与卫生统计学2021级，南京 211100
- Keywords:
Gene edit;
2019-nCoV;
Nucleic acid testing
- From:
Chinese Journal of Laboratory Medicine
2024;47(6):658-666
- CountryChina
- Language:Chinese
-
Abstract:
Objective:Based on the specific cleavage and non-specific "trans-cleavage" activities of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein(CRISPR/Cas13), we established a visually amplification-free rapid detection technique of 2019-nCoV nucleic acid. This technique is easily processed with a low detection limit and good specificity.Methods:According to the 2019-nCoV gene sequence, specific CRISPR RNAs were screened and designed by bioinformatics analysis, and then synthesized as universal signal-strained RNA transcription targets in vitro to establish and optimize the reaction system. Moreover, the 2019-nCoV pseudoviral nucleic acid was used as a standard substance to evaluate the detection limit. A total of 65 positive samples were collected from various 2019-nCoV variants, while 48 negative samples included other clinically common respiratory pathogens, such as influenza A virus, influenza B virus, human parainfluenza virus, Klebsiella pneumonia, etc. All samples were tested by quantitative PCR (qPCR), digital PCR, and the method established in this study. The sensitivity and specificity of the newly established method were analyzed and evaluated. Results:With the newly established technique, the detection time for 2019-nCoV nucleic acid could be minimized to 6 minutes. In addition, the detection limit was 14 copies/μl when assisted by the displaying instrument, whereas it increased to 28 copies/μl with the naked eye. This technique had a sensitivity and specificity of 98.5% (66/67) and 100% (46/46) respectively, showing no statistically significant difference compared to the gold standard qPCR( P=1). Conclusions:This study has successfully established a CRISPR/Cas13a-based visually rapid detection technique for 2019-nCoV nucleic acid. This technique offers the advantages of a simple process, convenient operation, low environmental operating requirements, a detection limit close to qPCR, and a strong potential for on-site testing applications.
