Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids
Name
2020.11.03.20223602v3.full.pdf
Description
Submitted version
Size
1.83 MB
Format
Adobe PDF
Checksum (MD5)
797b36fa1393001322118060fe6f76a1
Author(s)
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
Date Issued
2021
Journal
Biomaterials
Publisher
Elsevier BV
Citation
Wu, Xiaolin, Tay, Joshua K, Goh, Chuan Keng, Chan, Cheryl, Lee, Yie Hou et al. 2021. "Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids." Biomaterials, 274.
Version
Original manuscript
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.
MIT Department
Singapore-MIT Alliance in Research and Technology (SMART)
Massachusetts Institute of Technology. Center for Biomedical Innovation
Massachusetts Institute of Technology. Synthetic Biology Center
Massachusetts Institute of Technology. Research Laboratory of Electronics
Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science
Harvard University--MIT Division of Health Sciences and Technology
Massachusetts Institute of Technology. Department of Biological Engineering
Terms of Use
Creative Commons Attribution-NonCommercial-NoDerivs License
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DOI of Published Version
https://doi.org/10.1016/J.BIOMATERIALS.2021.120876
