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Volume 12, Issue 3 (Vol.12 No.3 Oct 2023)
rbmb.net 2023, 12(3): 425-437 |
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Li J, Wang X, Chen L, Duan L, Tan F, Li K et al . SARS-CoV‑2 and Its Omicron Variants Detection with RT-RPA -CRISPR/Cas13a-Based Method at Room Temperature. rbmb.net 2023; 12 (3) :425-437
URL: http://rbmb.net/article-1-1237-en.html
URL: http://rbmb.net/article-1-1237-en.html
Translational Medicine Institute, the First People's Hospital of Chenzhou, Hengyang Medical School, University of South China, Chenzhou 423000, Hunan, China & National Engineering Research Center of Personalized Diagnostic and Therapeutic Technology, Hunan University of Chinese Medicine, Changsha 410208, Hunan, China.
Abstract: (427 Views)
Background: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global health crisis, with genetic mutations and evolution further creating uncertainty about epidemic risk. It is imperative to rapidly determine the nucleic acid sequence of SARS-CoV-2 and its variants to combat the coronavirus pandemic. Our goal was to develop a rapid, room-temperature, point-of-care (POC) detection system to determine the nucleic acid sequences of SARS-CoV-2 isolates, especially omicron variants.
Methods: Based on the conserved nucleotide sequence of SARS-CoV-2, bioinformatics software was used to analyze, design, and screen optimal enzymatic isothermal amplification primers and efficient CRISPR RNAs (crRNAs) of CRISPR/Cas13a to the target sequences. Reverse transcription-recombinase polymerase amplification (RT-RPA) was used to amplify the virus, and CRISPR/Cas13a-crRNA was used to cleave the SARS-CoV-2 target sequence. The sensitivity of nucleic acid detection was assessed by serial dilution of plasmid templates. All reactions were performed at room temperature.
Results: RT-RPA, combined with CRISPR/Cas13a, can detect the SARS-CoV-2 with a minimum content of 102 copies/μL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 103 copies/μL.
Conclusion: The method developed in this study has potential application in clinical detection of SARS-CoV-2 and its omicron variants.
Methods: Based on the conserved nucleotide sequence of SARS-CoV-2, bioinformatics software was used to analyze, design, and screen optimal enzymatic isothermal amplification primers and efficient CRISPR RNAs (crRNAs) of CRISPR/Cas13a to the target sequences. Reverse transcription-recombinase polymerase amplification (RT-RPA) was used to amplify the virus, and CRISPR/Cas13a-crRNA was used to cleave the SARS-CoV-2 target sequence. The sensitivity of nucleic acid detection was assessed by serial dilution of plasmid templates. All reactions were performed at room temperature.
Results: RT-RPA, combined with CRISPR/Cas13a, can detect the SARS-CoV-2 with a minimum content of 102 copies/μL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 103 copies/μL.
Conclusion: The method developed in this study has potential application in clinical detection of SARS-CoV-2 and its omicron variants.
Type of Article: Original Article |
Subject:
Molecular Biology
Received: 2023/08/29 | Accepted: 2024/01/14 | Published: 2024/02/25
Received: 2023/08/29 | Accepted: 2024/01/14 | Published: 2024/02/25
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