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

Xiaolin Wu; Cheryl Chan; Stacy L Springs; Yie Hou Lee; Timothy K Lu; Hanry Yu

doi:10.1016/j.aca.2022.339494

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

Anal Chim Acta. 2022 Mar 1:1196:339494. doi: 10.1016/j.aca.2022.339494. Epub 2022 Jan 25.

Authors

Xiaolin Wu¹, Cheryl Chan¹, Stacy L Springs², Yie Hou Lee¹, Timothy K Lu³, Hanry Yu⁴

Affiliations

¹ Critical Analytics for Manufacturing Personalized Medicine Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, 138602, Singapore.
² Critical Analytics for Manufacturing Personalized Medicine Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, 138602, Singapore; Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
³ Critical Analytics for Manufacturing Personalized Medicine Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, 138602, Singapore; Synthetic Biology Center, Massachusetts Institute of Technology (MIT), Cambridge, MA, 02139, USA; Synthetic Biology Group, Research Laboratory of Electronics, Massachusetts Institute of Technology (MIT), Cambridge, MA, 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA; Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology (MIT), Cambridge, MA, 02142, USA; Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA, 02142, USA. Electronic address: timlu@mit.edu.
⁴ Critical Analytics for Manufacturing Personalized Medicine Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, 138602, Singapore; Institute of Bioengineering and Bioimaging (IBB), A∗STAR, the Nanos, #04-01, 31 Biopolis Way, 138669, Singapore; Mechanobiology Institute, National University of Singapore, T-Lab, #05-01, 5A Engineering Drive 1, 117411, Singapore; Department of Physiology & the Institute for Digital Medicine (WisDM), Yong Loo Lin School of Medicine, MD9-04-11, 2 Medical Drive, 117593, Singapore. Electronic address: phsyuh@nus.edu.sg.

PMID: 35151407
DOI: 10.1016/j.aca.2022.339494

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.

Keywords: CRISPR/Cas; Isothermal amplification; Nucleic acids detection and quantification; Virus detection and quantification; Warmstart digital CRISPR.

MeSH terms

COVID-19*
CRISPR-Cas Systems / genetics
Humans
Nucleic Acid Amplification Techniques
Nucleic Acids*
RNA, Viral / genetics
SARS-CoV-2
Sensitivity and Specificity

Substances

Nucleic Acids
RNA, Viral