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

被引：90

作者：

Chandrasekaran, Sita S. ^{[1
,2
,3
]}

Agrawal, Shreeya ^{[1
,2
]}

Fanton, Alison ^{[1
,2
,3
]}

Jangid, Aditya R. ^{[1
,2
]}

Charrez, Berenice ^{[3
]}

Escajeda, Arturo M. ^{[4
]}

Son, Sungmin ^{[1
]}

Mcintosh, Roger ^{[4
]}

Tran, Huyen ^{[4
]}

Bhuiya, Abdul ^{[1
,3
]}

Derby, Maria Diaz de Leon ^{[1
,3
]}

Switz, Neil A. ^{[5
]}

Armstrong, Maxim ^{[6
]}

Harris, Andrew R. ^{[1
]}

Prywes, Noam ^{[2
]}

Lukarska, Maria ^{[2
,7
]}

Biering, Scott B. ^{[8
]}

Smock, Dylan C. J. ^{[2
,7
]}

Mok, Amanda ^{[9
]}

Knott, Gavin J. ^{[2
,7
,10
]}

Dang, Qi ^{[2
]}

Van Dis, Erik ^{[7
]}

Dugan, Eli ^{[2
,7
]}

Kim, Shin ^{[2
,7
]}

Liu, Tina Y. ^{[2
,7
]}

Consortium, Igi Testing

Moehle, Erica A. ^{[2
]}

Kogut, Katherine ^{[11
]}

Eskenazi, Brenda ^{[11
]}

Harris, Eva ^{[8
]}

Stanley, Sarah A. ^{[7
,12
]}

Lareau, Liana F. ^{[1
,2
]}

Tan, Ming X. ^{[4
]}

Fletcher, Daniel A. ^{[1
]}

Doudna, Jennifer A. ^{[2
,6
,7
,13
,14
,15
]}

Savage, David F. ^{[7
]}

Hsu, Patrick D. ^{[1
,2
,16
]}

机构：

[1] Univ Calif Berkeley, Dept Bioengn, Berkeley, CA 94720 USA

[2] Univ Calif Berkeley, Innovat Genom Inst, Berkeley, CA 94720 USA

[3] Univ Calif Berkeley, Univ Calif San Francisco, Grad Program Bioengn, Berkeley, CA 94720 USA

[4] Wainamics Inc, Pleasanton, CA USA

[5] San Jose State Univ, Dept Phys & Astron, San Jose, CA 95192 USA

[6] Lawrence Berkeley Natl Lab, Mol Biophys & Integrated Bioimaging Div, Berkeley, CA 94720 USA

[7] Univ Calif Berkeley, Dept Mol & Cell Biol, 229 Stanley Hall, Berkeley, CA 94720 USA

[8] Univ Calif Berkeley, Sch Publ Hlth, Div Infect Dis & Vaccinol, Berkeley, CA 94720 USA

[9] Univ Calif Berkeley, Ctr Computat Biol, Berkeley, CA 94720 USA

[10] Monash Univ, Monash Biomed Discovery Inst, Dept Biochem & Mol Biol, Clayton, Vic, Australia

[11] Univ Calif Berkeley, Sch Publ Hlth, CERCH, Berkeley, CA 94720 USA

[12] Univ Calif Berkeley, Sch Publ Hlth, Berkeley, CA 94720 USA

[13] Univ Calif Berkeley, Howard Hughes Med Inst, Berkeley, CA 94720 USA

[14] Univ Calif Berkeley, Dept Chem, Berkeley, CA 94720 USA

[15] Gladstone Inst, Gladstone Inst Data Sci & Biotechnol, San Francisco, CA 94158 USA

[16] Arc Inst, Palo Alto, CA 94304 USA

来源：

NATURE BIOMEDICAL ENGINEERING | 2022年 / 6卷 / 08期

基金：

美国国家卫生研究院;

关键词：

NUCLEIC-ACID DETECTION; DIAGNOSTICS;

D O I：

10.1038/s41551-022-00917-y

中图分类号：

R318 [生物医学工程];

学科分类号：

0831 ;

摘要：

A COVID-19 test implemented in an automated microfluidic device and leveraging isothermal RNA amplification followed by T7 transcription and Cas13-mediated cleavage of a quenched fluorophore rapidly detects SARS-CoV-2 RNA in saliva samples. 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 mu l(-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.

引用

页码：944 / +

页数：20

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