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

Biomaterials. 2021 Jul;274:120876. doi: 10.1016/j.biomaterials.2021.120876. Epub 2021 May 11.

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 (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.

Keywords: Absolute quantification; CRISPR/Cas; Molecular diagnosis; Virus detection.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • COVID-19*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Epstein-Barr Virus Infections*
  • Herpesvirus 4, Human / genetics
  • Humans
  • Nucleic Acids*
  • SARS-CoV-2
  • Sensitivity and Specificity

Substances

  • Nucleic Acids