Rapid detection of SARS-CoV-2 RNA in saliva via Cas13

Sita S Chandrasekaran; Shreeya Agrawal; Alison Fanton; Aditya R Jangid; Bérénice Charrez; Arturo M Escajeda; Sungmin Son; Roger Mcintosh; Huyen Tran; Abdul Bhuiya; María Díaz de León Derby; Neil A Switz; Maxim Armstrong; Andrew R Harris; Noam Prywes; Maria Lukarska; Scott B Biering; Dylan C J Smock; Amanda Mok; Gavin J Knott; Qi Dang; Erik Van Dis; Eli Dugan; Shin Kim; Tina Y Liu; IGI Testing Consortium; Erica A Moehle; Katherine Kogut; Brenda Eskenazi; Eva Harris; Sarah A Stanley; Liana F Lareau; Ming X Tan; Daniel A Fletcher; Jennifer A Doudna; David F Savage; Patrick D Hsu

doi:10.1038/s41551-022-00917-y

Rapid detection of SARS-CoV-2 RNA in saliva via Cas13

Nat Biomed Eng. 2022 Aug;6(8):944-956. doi: 10.1038/s41551-022-00917-y. Epub 2022 Aug 11.

Authors

Sita S Chandrasekaran^#^{1

2

3}, Shreeya Agrawal^#^{1

2}, Alison Fanton^#^{1

2

3}, Aditya R Jangid^#^{1

2}, Bérénice Charrez³, Arturo M Escajeda⁴, Sungmin Son¹, Roger Mcintosh⁴, Huyen Tran⁴, Abdul Bhuiya^{1

3}, María Díaz de León Derby^{1

3}, Neil A Switz⁵, Maxim Armstrong⁶, Andrew R Harris¹, Noam Prywes², Maria Lukarska^{2

7}, Scott B Biering⁸, Dylan C J Smock^{2

7}, Amanda Mok⁹, Gavin J Knott^{2

7

10}, Qi Dang², Erik Van Dis⁷, Eli Dugan^{2

7}, Shin Kim^{2

7}, Tina Y Liu^{2

7}; IGI Testing Consortium; Erica A Moehle², Katherine Kogut¹¹, Brenda Eskenazi¹¹, Eva Harris⁸, Sarah A Stanley^{7

12}, Liana F Lareau^{1

2}, Ming X Tan⁴, Daniel A Fletcher¹, Jennifer A Doudna^{13

14

15

16

17

18}, David F Savage¹⁹, Patrick D Hsu^{20

21

22}

Collaborators

IGI Testing Consortium:
Jennifer R Hamilton, Enrique Lin-Shiao, Elizabeth C Stahl, Connor A Tsuchida, Petros Giannikopoulos, Matthew McElroy, Shana McDevitt, Arielle Zur, Iman Sylvain, Alison Ciling, Madeleine Zhu, Clara Williams, Alisha Baldwin

Affiliations

¹ Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
² Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
³ University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, Berkeley, CA, USA.
⁴ Wainamics Inc., Pleasanton, CA, USA.
⁵ Department of Physics and Astronomy, San José State University, San José, CA, USA.
⁶ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
⁷ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
⁸ Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, Berkeley, CA, USA.
⁹ Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA.
¹⁰ Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
¹¹ Center for Environmental Research and Community Health (CERCH), School of Public Health, University of California, Berkeley, Berkeley, CA, USA.
¹² School of Public Health, University of California, Berkeley, Berkeley, CA, USA.
¹³ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁴ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁵ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁶ Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁷ Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁸ Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA. doudna@berkeley.edu.
¹⁹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA. savage@berkeley.edu.
²⁰ Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.
²¹ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.
²² Arc Institute, Palo Alto, CA, USA. pdhsu@berkeley.edu.

^# Contributed equally.

Abstract

Rapid nucleic acid testing is central to infectious disease surveillance. Here, we report an assay for rapid COVID-19 testing and its implementation in a prototype microfluidic device. The assay, which we named DISCoVER (for diagnostics with coronavirus enzymatic reporting), involves extraction-free sample lysis via shelf-stable and low-cost reagents, multiplexed isothermal RNA amplification followed by T7 transcription, and Cas13-mediated cleavage of a quenched fluorophore. The device consists of a single-use gravity-driven microfluidic cartridge inserted into a compact instrument for automated running of the assay and readout of fluorescence within 60 min. DISCoVER can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva with a sensitivity of 40 copies μl^-1, and was 94% sensitive and 100% specific when validated (against quantitative PCR) using total RNA extracted from 63 nasal-swab samples (33 SARS-CoV-2-positive, with cycle-threshold values of 13-35). The device correctly identified all tested clinical saliva samples (10 SARS-CoV-2-positive out of 13, with cycle-threshold values of 23-31). Rapid point-of-care nucleic acid testing may broaden the use of molecular diagnostics.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

COVID-19 Testing
COVID-19* / diagnosis
Humans
RNA, Viral / genetics
SARS-CoV-2* / genetics
Saliva

Substances

RNA, Viral

Abstract

Publication types

MeSH terms

Substances

Grants and funding