tetano
Editor, Senior Moderator
J Med Virol
. 2022 Aug 27.
doi: 10.1002/jmv.28096. Online ahead of print.
Highly sensitive and rapid detection of SARS-CoV-2 via a portable CRISPR-Cas13a-based lateral flow assay
Hongbo Liu[SUP] 1 [/SUP], Shuailei Chang[SUP] 1 2 [/SUP], Sijia Chen[SUP] 1 [/SUP], Yue Du[SUP] 1 2 [/SUP], Hui Wang[SUP] 1 [/SUP], Chao Wang[SUP] 1 [/SUP], Ying Xiang[SUP] 1 [/SUP], Qi Wang[SUP] 1 [/SUP], Zhenjun Li[SUP] 3 [/SUP], Shengqi Wang[SUP] 4 [/SUP], Shaofu Qiu[SUP] 1 [/SUP], Hongbin Song[SUP] 1 [/SUP]
Affiliations
Abstract
To rapidly identify individuals infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and control the spread of coronavirus disease (COVID-19), there is an urgent need for highly sensitive on-site virus detection methods. A clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)-based molecular diagnostic method was developed for this purpose. Here, a CRISPR system-mediated lateral flow assay (LFA) for SARS-CoV-2 was established based on multienzyme isothermal rapid amplification, CRISPR-Cas13a nuclease, and LFA. To improve the limit of detection, the crRNA, amplification primer, and probe were screened, in addition to concentrations of various components in the reaction system. The limit of detection of CRISPR detection was improved to 0.25 copy/μL in both fluorescence- and immunochromatography-based assays. To enhance the quality control of the CRISPR-based LFA method, GAPDH was detected as a reference using a triple-line strip design in a lateral flow strip. In total, 52 COVID-19-positive and 101 COVID-19-negative clinical samples examined by RT-PCR were tested using the CRISPR immunochromatographic detection technique. Results revealed 100% consistency, indicating the comparable effectiveness of our method to that of RT-PCR. In conclusion, this approach significantly improves the sensitivity and reliability of CRISPR-mediated LFA and provides a crucial tool for on-site detection of SARS-CoV-2. This article is protected by copyright. All rights reserved.
Keywords: CRISPR; SARS-CoV-2; lateral flow assay; sensitive detection.
. 2022 Aug 27.
doi: 10.1002/jmv.28096. Online ahead of print.
Highly sensitive and rapid detection of SARS-CoV-2 via a portable CRISPR-Cas13a-based lateral flow assay
Hongbo Liu[SUP] 1 [/SUP], Shuailei Chang[SUP] 1 2 [/SUP], Sijia Chen[SUP] 1 [/SUP], Yue Du[SUP] 1 2 [/SUP], Hui Wang[SUP] 1 [/SUP], Chao Wang[SUP] 1 [/SUP], Ying Xiang[SUP] 1 [/SUP], Qi Wang[SUP] 1 [/SUP], Zhenjun Li[SUP] 3 [/SUP], Shengqi Wang[SUP] 4 [/SUP], Shaofu Qiu[SUP] 1 [/SUP], Hongbin Song[SUP] 1 [/SUP]
Affiliations
- PMID: 36029033
- DOI: 10.1002/jmv.28096
Abstract
To rapidly identify individuals infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and control the spread of coronavirus disease (COVID-19), there is an urgent need for highly sensitive on-site virus detection methods. A clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)-based molecular diagnostic method was developed for this purpose. Here, a CRISPR system-mediated lateral flow assay (LFA) for SARS-CoV-2 was established based on multienzyme isothermal rapid amplification, CRISPR-Cas13a nuclease, and LFA. To improve the limit of detection, the crRNA, amplification primer, and probe were screened, in addition to concentrations of various components in the reaction system. The limit of detection of CRISPR detection was improved to 0.25 copy/μL in both fluorescence- and immunochromatography-based assays. To enhance the quality control of the CRISPR-based LFA method, GAPDH was detected as a reference using a triple-line strip design in a lateral flow strip. In total, 52 COVID-19-positive and 101 COVID-19-negative clinical samples examined by RT-PCR were tested using the CRISPR immunochromatographic detection technique. Results revealed 100% consistency, indicating the comparable effectiveness of our method to that of RT-PCR. In conclusion, this approach significantly improves the sensitivity and reliability of CRISPR-mediated LFA and provides a crucial tool for on-site detection of SARS-CoV-2. This article is protected by copyright. All rights reserved.
Keywords: CRISPR; SARS-CoV-2; lateral flow assay; sensitive detection.