Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids.
Biomaterials
; 274: 120876, 2021 07.
Article
in English
| MEDLINE | ID: covidwho-1225152
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
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Nucleic Acids
/
Epstein-Barr Virus Infections
/
COVID-19
Type of study:
Diagnostic study
/
Prognostic study
/
Randomized controlled trials
Limits:
Humans
Language:
English
Journal:
Biomaterials
Year:
2021
Document Type:
Article
Affiliation country:
J.biomaterials.2021.120876
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