A Rapid DIgital Crispr Approach (RADICA) for the detection and absolute quantification of nucleic acids

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 and Epstein-Barr virus (EBV) as a proof-of-concept target, RADICA allows for absolute quantification with high accuracy and low variability, no cross-reactivity to similar targets, and high tolerance to human background DNA. RADICA therefore enables rapid and sensitive absolute quantification of nucleic acids which can be widely applied across clinical, research, and biomanufacturing areas.