Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids. (July 2021)

• Record Type: Journal Article
• 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
• Is Part Of: Biomaterials. Volume 274(2021)
• Publication Date: 2021-07
• Subjects: CRISPR/Cas -- Absolute quantification -- Molecular diagnosis -- Virus detection; Biomedical materials -- Periodicals; Biocompatible Materials -- Periodicals; Biomatériaux -- Périodiques; 610.28
• DOI: 10.1016/j.biomaterials.2021.120876
• Languages: English
• ISSNs: 0142-9612

Abstract

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 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 (R 2 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 (R 2 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.