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

Author(s)
Wu, Xiaolin; Chan, Cheryl; Springs, Stacy L; Lee, Yie Hou; Lu, Timothy K; Yu, Hanry; ... Show more Show less
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Abstract
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/μl 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/μL for DNA and 1.2-18391 copies/μL for RNA (both R2 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.
Date issued
2022
URI
https://hdl.handle.net/1721.1/143766
Department
Massachusetts Institute of Technology. Synthetic Biology Center; Singapore-MIT Alliance in Research and Technology (SMART); 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; Massachusetts Institute of Technology. Department of Biological Engineering
Journal
Analytica Chimica Acta
Publisher
Elsevier BV
Citation
Wu, Xiaolin, Chan, Cheryl, Springs, Stacy L, Lee, Yie Hou, Lu, Timothy K et al. 2022. "A warm-start digital CRISPR/Cas-based method for the quantitative detection of nucleic acids." Analytica Chimica Acta, 1196.
Version: Original manuscript

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