tetano
Editor, Senior Moderator
Front Microbiol
. 2022 Jun 28;13:945133.
doi: 10.3389/fmicb.2022.945133. eCollection 2022.
Combination of Isothermal Recombinase-Aided Amplification and CRISPR-Cas12a-Mediated Assay for Rapid Detection of Major Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern
Hongqing Lin[SUP] 1 [/SUP], Yuanhao Liang[SUP] 1 [/SUP], Lirong Zou[SUP] 2 [/SUP], Baisheng Li[SUP] 2 [/SUP], Jianhui Zhao[SUP] 1 [/SUP], Haiying Wang[SUP] 1 [/SUP], Jiufeng Sun[SUP] 2 [/SUP], Xiaoling Deng[SUP] 2 [/SUP], Shixing Tang[SUP] 1 3 4 [/SUP]
Affiliations
Abstract
Coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 variants is a new and unsolved threat; therefore, it is an urgent and unmet need to develop a simple and rapid method for detecting and tracking SARS-CoV-2 variants. The spike gene of SARS-CoV-2 was amplified by isothermal recombinase-aided amplification (RAA) followed by the cleavage of CRISPR-Cas12a in which five allele-specific crRNAs and two Omicron-specific crRNAs were designed to detect and distinguish major SARS-CoV-2 variants of concerns (VOCs), including alpha, beta, delta variants, and Omicron sublineages BA.1 and BA.2. The whole reaction can be carried out in one tube at 39°C within 1.5-2 h, and the results can be read out by a fluorescence meter or naked eyes. Our results show that the RAA/CRISPR-Cas12a-based assay could readily distinguish the signature mutations, i.e., K417N, T478K, E484K, N501Y, and D614G, with a sensitivity of 100.0% and a specificity of 94.9-100.0%, respectively. The assay had a low limit of detection (LOD) of 10[SUP]4[/SUP] copies/reaction and a concordance of 92.59% with Sanger sequencing results when detecting 54 SARS-CoV-2 positive clinical samples. The two Omicron-specific crRNAs can readily and correctly distinguish Omicron BA.1 and BA.2 sublineages with a LOD of as low as 20 copies/reaction. Furthermore, no cross-reaction was observed for all crRNAs analyzed when detecting clinical samples infected with 11 common respiratory pathogens. The combination of isothermal amplification and CRISPR-Cas12a-mediated assay is suitable for rapid detection of major SARS-CoV-2 variants in point-of-care testing and in resource-limiting settings. This simple assay could be quickly updated for emerging variants and implemented to routinely monitor and track the spread of SARS-CoV-2 variants.
Keywords: CRISPR-Cas12a; SARS-CoV-2; isothermal amplification; variant genotyping; variants of concern.
. 2022 Jun 28;13:945133.
doi: 10.3389/fmicb.2022.945133. eCollection 2022.
Combination of Isothermal Recombinase-Aided Amplification and CRISPR-Cas12a-Mediated Assay for Rapid Detection of Major Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern
Hongqing Lin[SUP] 1 [/SUP], Yuanhao Liang[SUP] 1 [/SUP], Lirong Zou[SUP] 2 [/SUP], Baisheng Li[SUP] 2 [/SUP], Jianhui Zhao[SUP] 1 [/SUP], Haiying Wang[SUP] 1 [/SUP], Jiufeng Sun[SUP] 2 [/SUP], Xiaoling Deng[SUP] 2 [/SUP], Shixing Tang[SUP] 1 3 4 [/SUP]
Affiliations
- PMID: 35836420
- PMCID: PMC9274097
- DOI: 10.3389/fmicb.2022.945133
Abstract
Coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 variants is a new and unsolved threat; therefore, it is an urgent and unmet need to develop a simple and rapid method for detecting and tracking SARS-CoV-2 variants. The spike gene of SARS-CoV-2 was amplified by isothermal recombinase-aided amplification (RAA) followed by the cleavage of CRISPR-Cas12a in which five allele-specific crRNAs and two Omicron-specific crRNAs were designed to detect and distinguish major SARS-CoV-2 variants of concerns (VOCs), including alpha, beta, delta variants, and Omicron sublineages BA.1 and BA.2. The whole reaction can be carried out in one tube at 39°C within 1.5-2 h, and the results can be read out by a fluorescence meter or naked eyes. Our results show that the RAA/CRISPR-Cas12a-based assay could readily distinguish the signature mutations, i.e., K417N, T478K, E484K, N501Y, and D614G, with a sensitivity of 100.0% and a specificity of 94.9-100.0%, respectively. The assay had a low limit of detection (LOD) of 10[SUP]4[/SUP] copies/reaction and a concordance of 92.59% with Sanger sequencing results when detecting 54 SARS-CoV-2 positive clinical samples. The two Omicron-specific crRNAs can readily and correctly distinguish Omicron BA.1 and BA.2 sublineages with a LOD of as low as 20 copies/reaction. Furthermore, no cross-reaction was observed for all crRNAs analyzed when detecting clinical samples infected with 11 common respiratory pathogens. The combination of isothermal amplification and CRISPR-Cas12a-mediated assay is suitable for rapid detection of major SARS-CoV-2 variants in point-of-care testing and in resource-limiting settings. This simple assay could be quickly updated for emerging variants and implemented to routinely monitor and track the spread of SARS-CoV-2 variants.
Keywords: CRISPR-Cas12a; SARS-CoV-2; isothermal amplification; variant genotyping; variants of concern.