tetano
Editor, Senior Moderator
Rep Biochem Mol Biol
. 2023 Oct;12(3):425-437.
doi: 10.61186/rbmb.12.3.425. SARS-CoV-2 and Its Omicron Variants Detection with RT-RPA -CRISPR/Cas13a-Based Method at Room Temperature
Jia Li[SUP] 1 2 3 [/SUP], Xiaojun Wang[SUP] 1 2 4 [/SUP], Liujie Chen[SUP] 2 3 [/SUP], Lili Duan[SUP] 2 3 [/SUP], Fenghua Tan[SUP] 2 3 [/SUP], Kai Li[SUP] 3 5 [/SUP], Zheng Hu[SUP] 2 3 [/SUP]
Affiliations
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 10[SUP]2[/SUP] copies/μL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 10[SUP]3[/SUP] copies/μL.
Conclusions: 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.
. 2023 Oct;12(3):425-437.
doi: 10.61186/rbmb.12.3.425. SARS-CoV-2 and Its Omicron Variants Detection with RT-RPA -CRISPR/Cas13a-Based Method at Room Temperature
Jia Li[SUP] 1 2 3 [/SUP], Xiaojun Wang[SUP] 1 2 4 [/SUP], Liujie Chen[SUP] 2 3 [/SUP], Lili Duan[SUP] 2 3 [/SUP], Fenghua Tan[SUP] 2 3 [/SUP], Kai Li[SUP] 3 5 [/SUP], Zheng Hu[SUP] 2 3 [/SUP]
Affiliations
- PMID: 38618265
- PMCID: PMC11015926
- DOI: 10.61186/rbmb.12.3.425
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 10[SUP]2[/SUP] copies/μL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 10[SUP]3[/SUP] copies/μL.
Conclusions: 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.