Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids

• Published in: Biomaterials 2021-07, Vol.274, p.120876-120876, Article 120876
• Main Authors: Wu, Xiaolin, Tay, Joshua K., Goh, Chuan Keng, Chan, Cheryl, Lee, Yie Hou, Springs, Stacy L., Wang, De Yun, Loh, Kwok Seng, Lu, Timothy K., Yu, Hanry
• Format: Article
• Language: English
• Subjects: Absolute quantification; CRISPR/Cas; Molecular diagnosis; Virus detection
• ISSN: 0142-9612; 1878-5905
• DOI: 10.1016/j.biomaterials.2021.120876

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 1 h 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 in 40-60 min. Using SARS-CoV-2 as a proof-of-concept target, RADICA allows for absolute quantification with a linear dynamic range of 0.6–2027 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 0.5–2100 copies/μL (R2 value > 0.98) in 1 h without thermal cycling, providing a 4-fold faster alternative to digital PCR-based detection. RADICA therefore enables rapid and sensitive absolute quantification of nucleic acids which can be widely applied across clinical, research, and biomanufacturing areas.