A warm-start digital CRISPR/Cas-based method for the quantitative detection of nucleic acids

Nucleic acids-based molecular diagnostic tools incorporating the CRISPR/Cas system are being developed as rapid and sensitive methods for pathogen detection. However, most CRISPR/Cas-based diagnostics lack quantitative detection ability. Here, we report Warm-Start RApid Digital Crispr Approach (WS-RADICA) for the rapid, sensitive, and quantitative detection of nucleic acids. WS-RADICA detected as little as 1 copy/mu l SARS-CoV-2 RNA in 40 min (qualitative detection) or 60 min (quantitative detection). WS-RADICA can be easily adapted to various digital devices: two digital chips were evaluated for both DNA and RNA quantification, with linear dynamic ranges of 0.8-12777 copies/mu L for DNA and 1.2-18391 copies/mu L for RNA (both R-2 values > 0.99). Moreover, WS-RADICA had lower detection limit and higher inhibitor tolerance than a bulk RT-LAMP-Cas12b reaction and similar performance to RT-qPCR and RT-dPCR. To prove its performance on nucleic acids derived from live virus, WS-RADICA was also validated to detect and quantify human adenovirus and herpes simplex virus. Given its speed, sensitivity, quantification capability, and inhibitor tolerance, WS-RADICA shows great promise for a variety of applications requiring nucleic acid quantification. (C) 2022 Elsevier B.V. All rights reserved.

Automated summary

WS-RADICA is a new nucleic acid detection method that enables rapid, sensitive, and quantitative detection of nucleic acids. It has a lower detection limit and higher inhibitor tolerance, and has been validated for nucleic acids derived from live viruses.