Digital CRISPR-based Method for the Rapid Detection and Absolute Quantification of Nucleic Acids

2021 
Abstract Rapid diagnostics of adventitious agents in biopharmaceutical/cell manufacturing release testing and the fight against viral infection have become critical. Quantitative real-time PCR and CRISPR-based methods rapidly detect DNA/RNA in one hour (1h) but suffer from inter-site variability. Absolute quantification of DNA/RNA by methods such as digital PCR reduce this variability but are currently too slow for wider application. Here, we report a RApid DIgital Crispr Approach (RADICA) for absolute quantification of nucleic acids within 1h. Using SARS-CoV-2 as a proof-of-concept target, RADICA allows for absolute quantification with a linear dynamic range of 0.6 to 2027 copies/μL (R2 value > 0.99), high accuracy and low variability, no cross-reactivity to similar targets, and high tolerance to human background DNA. RADICA’s versatility is validated against other targets such as Epstein-Barr virus (EBV) from human B cells and patients’ serum. RADICA can accurately detect and absolutely quantify EBV DNA with similar dynamic range of 0.5 to 2100 copies/μL (R2 value > 0.98) in 1h without thermal cycling, providing a 4-fold faster alternative to digital PCR-based detection. RADICA therefore enables rapid and sensitive absolute quantification of DNA/RNA which can be widely applied across clinical, research, and biomanufacturing areas.
    • Correction
    • Source
    • Cite
    • Save
    • Machine Reading By IdeaReader
    73
    References
    7
    Citations
    NaN
    KQI
    []