SARS-CoV‑۲ and Its Omicron Variants Detection with RT-RPA -CRISPR/Cas۱۳a-Based Method at Room Temperature

Background: The outbreak of severe acute respiratory syndrome coronavirus ۲ (SARS-CoV-۲) 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-۲ 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-۲ isolates, especially omicron variants. Methods: Based on the conserved nucleotide sequence of SARS-CoV-۲, bioinformatics software was used to analyze, design, and screen optimal enzymatic isothermal amplification primers and efficient CRISPR RNAs (crRNAs) of CRISPR/Cas۱۳a to the target sequences. Reverse transcription-recombinase polymerase amplification (RT-RPA) was used to amplify the virus, and CRISPR/Cas۱۳a-crRNA was used to cleave the SARS-CoV-۲ 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/Cas۱۳a, can detect the SARS-CoV-۲ with a minimum content of ۱۰۲ copies/μL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of ۱۰۳ copies/μL. Conclusion: The method developed in this study has potential application in clinical detection of SARS-CoV-۲ and its omicron variants.

Keywords:

CRISPR/Cas۱۳a , Omicron variants , RT-RPA , SARS-CoV‑۲.