Title of article
SARS-CoV‑2 and Its Omicron Variants Detection with RT-RPA -CRISPR/Cas13a-Based Method at Room Temperature
Author/Authors
Li ، Jia First People s Hospital of Chenzhou, First Clinical College of Xiangnan University - Xiangnan University , Wang ، Xiaojun First Clinical College of Xiangnan University, First People s Hospital of Chenzhou - Xiangnan University , Chen ، Liujie First Clinical College of Xiangnan University, First People s Hospital of Chenzhou - Xiangnan University , Duan ، Lili First Clinical College of Xiangnan University, First People s Hospital of Chenzhou - Xiangnan University , Tan ، Fenghua First Clinical College of Xiangnan University, First People s Hospital of Chenzhou - Xiangnan University , Li ، Kai Translational Medicine Institute, First People s Hospital of Chenzhou, Hengyang Medical School - University of South China , Hu ، Zheng First Clinical College of Xiangnan University, First People s Hospital of Chenzhou - Xiangnan University
From page
425
To page
437
Abstract
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.
Keywords
CRISPR , Cas13a , Omicron variants , RT , RPA , SARS , CoV‑2
Journal title
Reports of Biochemistry and Molecular Biology (RBMB)
Journal title
Reports of Biochemistry and Molecular Biology (RBMB)
Record number
2759179
Link To Document