The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/μL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.
SUMMARY The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic and pre-symptomatic carriers of the virus. CRISPR-based diagnostics that utilize RNA and DNA-targeting enzymes can augment gold-standard PCR-based testing if they can be made rapid, portable and accurate. Here we report the development of an amplification-free CRISPR-Cas13a-based mobile phone assay for direct detection of SARS-CoV-2 from nasal swab RNA extracts. The assay achieved ∼100 copies/μL sensitivity in under 30 minutes and accurately detected a set of positive clinical samples in under 5 minutes. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity, and we directly quantified viral load using enzyme kinetics. Combined with mobile phone-based quantification, this assay can provide rapid, low-cost, point-of-care screening to aid in the control of SARS-CoV-2.
Carley N Gray合作论文数2Katherine S. Pollard合作论文数2Stephanie I Stephens合作论文数2María Díaz de León Derby合作论文数2Daniela Boehm合作论文数2
Abdul Bhuiya合作论文数University of California, Berkeley-University of California2Maira Phelps合作论文数2Anke Meyer-Franke合作论文数2
Andrew R Harris合作论文数Department of Mechanical and Aerospace Engineering, Carleton University2Jeffrey Shu合作论文数Gladstone Institutes2
Parinaz Fozouni合作论文数Gladstone Institutes, University of California, San Francisco, San Francisco, CA 94158, USA2
Gavin J. Knott合作论文数Univ Western Australia, Sch Chem & Biochem, Crawley, WA 6009, Australia2
Andreas Puschnik合作论文数Vir Biotechnology, Inc.2
ChunYu Zhao合作论文数Dalian University of Technology, Dalian Univ. of Technol.2
Emily Crawford合作论文数Delve Bio2D'Ambrosio Michael V合作论文数2
Gagandeep Renuka Kumar合作论文数Gladstone Institutes;University of California;Gladstone Institutes, University of California2
Jennifer A. Doudna合作论文数Department of Chemistry, College of Chemistry, University of California, Berkeley;Department of Molecular and Cell Biology, College of Letters & Science, University of California, Berkeley;Innovative Genomics Institute, University of California, Berkeley2
Daniel A. Fletcher合作论文数Lawrence Berkeley National Laboratory, University of California, Berkeley;Department of Bioengineering, College of Engineering, University of California, Berkeley;Fletcher Lab, University of California, Berkeley2Melanie Ott合作论文数Gladstone Institute of Virology and Immunology, University of California2
Chaz Langelier合作论文数Department of Medicine, School of Medicine, University of California, San Francisco2Neil A. Switz合作论文数2Amy Kistler合作论文数2Sungmin Son合作论文数College of Medicine, Seoul National University2
Chia-Lin Tsou合作论文数1Bastian Joehnk合作论文数1Keith Walcott合作论文数1Maxim Armstrong合作论文数1Pei-Yi Chen合作论文数1
Anita Sil合作论文数Department of Microbiology and Immunology, School of Medicine, University of California San Francisco1Joseph L. Derisi合作论文数1
Joe Derisi合作论文数Department of Biochemistry and Biophysics, School of Medicine, University of California, San Francisco1
