Reports of Biochemistry and Molecular Biology

en SARS-CoV‑2 and Its Omicron Variants Detection with RT-RPA -CRISPR/Cas13a-Based Method at Room Temperature زیست شناسی ملکولی Molecular Biology مقالات اصلی Original Article <div style="text-align: justify;">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.  </div> CRISPR/Cas13a, Omicron variants, RT-RPA, SARS-CoV‑2. 425 437 http://rbmb.net/browse.php?a_code=A-10-1346-1&slc_lang=en&sid=1 Jia Li 100319475328460018580 100319475328460018580 No The First Clinical College of Xiangnan University, the First People's Hospital of Chenzhou Affiliated Xiangnan University, Chenzhou 423000, Hunan, China & Translational Medicine Institute, the First People's Hospital of Chenzhou, Hengyang Medical School, University of South China, Chenzhou 423000, Hunan, China. Xiaojun Wang 100319475328460018581 100319475328460018581 No The First Clinical College of Xiangnan University, the First People's Hospital of Chenzhou Affiliated Xiangnan University, Chenzhou 423000, Hunan, China & The Oncology Department of the First People's Hospital of Chenzhou, Chenzhou 423000, Hunan, China. Liujie Chen 100319475328460018582 100319475328460018582 No The First Clinical College of Xiangnan University, the First People's Hospital of Chenzhou Affiliated Xiangnan University, Chenzhou 423000, Hunan, China & Translational Medicine Institute, the First People's Hospital of Chenzhou, Hengyang Medical School, University of South China, Chenzhou 423000, Hunan, China. Lili Duan 100319475328460018583 100319475328460018583 No The First Clinical College of Xiangnan University, the First People's Hospital of Chenzhou Affiliated Xiangnan University, Chenzhou 423000, Hunan, China & Translational Medicine Institute, the First People's Hospital of Chenzhou, Hengyang Medical School, University of South China, Chenzhou 423000, Hunan, China. Fenghua Tan 100319475328460018584 100319475328460018584 No The First Clinical College of Xiangnan University, the First People's Hospital of Chenzhou Affiliated Xiangnan University, Chenzhou 423000, Hunan, China & Translational Medicine Institute, the First People's Hospital of Chenzhou, Hengyang Medical School, University of South China, Chenzhou 423000, Hunan, China. Kai Li 100319475328460018585 100319475328460018585 Yes 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. Zheng Hu hu48005@163.com 100319475328460018586 100319475328460018586 No The First Clinical College of Xiangnan University, the First People's Hospital of Chenzhou Affiliated Xiangnan University, Chenzhou 423000, Hunan, China & Translational Medicine Institute, the First People's Hospital of Chenzhou, Hengyang Medical School, University of South China, Chenzhou 423000, Hunan, China.