Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA

Maturada Patchsung; Krittapas Jantarug; Archiraya Pattama; Kanokpol Aphicho; Surased Suraritdechachai; Piyachat Meesawat; Khomkrit Sappakhaw; Nattawat Leelahakorn; Theerawat Ruenkam; Thanakrit Wongsatit; Niracha Athipanyasilp; Bhumrapee Eiamthong; Benya Lakkanasirorat; Thitima Phoodokmai; Nootaree Niljianskul; Danaya Pakotiprapha; Sittinan Chanarat; Aimorn Homchan; Ruchanok Tinikul; Philaiwarong Kamutira; Kochakorn Phiwkaow; Sahachat Soithongcharoen; Chadaporn Kantiwiriyawanitch; Vinutsada Pongsupasa; Duangthip Trisrivirat; Juthamas Jaroensuk; Thanyaporn Wongnate; Somchart Maenpuen; Pimchai Chaiyen; Sirichai Kamnerdnakta; Jirawat Swangsri; Suebwong Chuthapisith; Yongyut Sirivatanauksorn; Chutikarn Chaimayo; Ruengpung Sutthent; Wannee Kantakamalakul; Julia Joung; Alim Ladha; Xin Jin; Jonathan S. Gootenberg; Omar O. Abudayyeh; Feng Zhang; Navin Horthongkham; Chayasith Uttamapinant

doi:10.1038/s41551-020-00603-x

Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA

Maturada Patchsung
, Krittapas Jantarug
, Archiraya Pattama
, Kanokpol Aphicho
, Surased Suraritdechachai
, Piyachat Meesawat
, Khomkrit Sappakhaw
, Nattawat Leelahakorn
, Theerawat Ruenkam
, Thanakrit Wongsatit
, Niracha Athipanyasilp
, Bhumrapee Eiamthong
, Benya Lakkanasirorat
, Thitima Phoodokmai
, Nootaree Niljianskul
, Danaya Pakotiprapha
, Sittinan Chanarat
, Aimorn Homchan
, Ruchanok Tinikul
, Philaiwarong Kamutira

Kochakorn Phiwkaow, Sahachat Soithongcharoen, Chadaporn Kantiwiriyawanitch, Vinutsada Pongsupasa, Duangthip Trisrivirat, Juthamas Jaroensuk, Thanyaporn Wongnate, Somchart Maenpuen, Pimchai Chaiyen, Sirichai Kamnerdnakta, Jirawat Swangsri, Suebwong Chuthapisith, Yongyut Sirivatanauksorn, Chutikarn Chaimayo, Ruengpung Sutthent, Wannee Kantakamalakul, Julia Joung, Alim Ladha, Xin Jin, Jonathan S. Gootenberg, Omar O. Abudayyeh, Feng Zhang, Navin Horthongkham^*, Chayasith Uttamapinant^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

551 Scopus citations

Abstract

Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)—the virus that causes coronavirus disease 2019 (COVID-19)—in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.

Original language	English
Pages (from-to)	1140-1149
Number of pages	10
Journal	Nature Biomedical Engineering
Volume	4
Issue number	12
DOIs	https://doi.org/10.1038/s41551-020-00603-x
State	Published - Dec 2020
Externally published	Yes

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Patchsung, M., Jantarug, K., Pattama, A., Aphicho, K., Suraritdechachai, S., Meesawat, P., Sappakhaw, K., Leelahakorn, N., Ruenkam, T., Wongsatit, T., Athipanyasilp, N., Eiamthong, B., Lakkanasirorat, B., Phoodokmai, T., Niljianskul, N., Pakotiprapha, D., Chanarat, S., Homchan, A., Tinikul, R., ... Uttamapinant, C. (2020). Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nature Biomedical Engineering, 4(12), 1140-1149. https://doi.org/10.1038/s41551-020-00603-x

@article{b87067e94daa4e70af22d3c5ba523936,

title = "Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA",

abstract = "Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)—the virus that causes coronavirus disease 2019 (COVID-19)—in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100\% specific and 100\% sensitive with a fluorescence readout, and 100\% specific and 97\% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100\% specific and 96\% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100\% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.",

author = "Maturada Patchsung and Krittapas Jantarug and Archiraya Pattama and Kanokpol Aphicho and Surased Suraritdechachai and Piyachat Meesawat and Khomkrit Sappakhaw and Nattawat Leelahakorn and Theerawat Ruenkam and Thanakrit Wongsatit and Niracha Athipanyasilp and Bhumrapee Eiamthong and Benya Lakkanasirorat and Thitima Phoodokmai and Nootaree Niljianskul and Danaya Pakotiprapha and Sittinan Chanarat and Aimorn Homchan and Ruchanok Tinikul and Philaiwarong Kamutira and Kochakorn Phiwkaow and Sahachat Soithongcharoen and Chadaporn Kantiwiriyawanitch and Vinutsada Pongsupasa and Duangthip Trisrivirat and Juthamas Jaroensuk and Thanyaporn Wongnate and Somchart Maenpuen and Pimchai Chaiyen and Sirichai Kamnerdnakta and Jirawat Swangsri and Suebwong Chuthapisith and Yongyut Sirivatanauksorn and Chutikarn Chaimayo and Ruengpung Sutthent and Wannee Kantakamalakul and Julia Joung and Alim Ladha and Xin Jin and Gootenberg, \{Jonathan S.\} and Abudayyeh, \{Omar O.\} and Feng Zhang and Navin Horthongkham and Chayasith Uttamapinant",

note = "Publisher Copyright: {\textcopyright} 2020, The Author(s), under exclusive licence to Springer Nature Limited.",

year = "2020",

month = dec,

doi = "10.1038/s41551-020-00603-x",

language = "英语",

volume = "4",

pages = "1140--1149",

journal = "Nature Biomedical Engineering",

issn = "2157-846X",

publisher = "Nature Research",

number = "12",

}

Patchsung, M, Jantarug, K, Pattama, A, Aphicho, K, Suraritdechachai, S, Meesawat, P, Sappakhaw, K, Leelahakorn, N, Ruenkam, T, Wongsatit, T, Athipanyasilp, N, Eiamthong, B, Lakkanasirorat, B, Phoodokmai, T, Niljianskul, N, Pakotiprapha, D, Chanarat, S, Homchan, A, Tinikul, R, Kamutira, P, Phiwkaow, K, Soithongcharoen, S, Kantiwiriyawanitch, C, Pongsupasa, V, Trisrivirat, D, Jaroensuk, J, Wongnate, T, Maenpuen, S, Chaiyen, P, Kamnerdnakta, S, Swangsri, J, Chuthapisith, S, Sirivatanauksorn, Y, Chaimayo, C, Sutthent, R, Kantakamalakul, W, Joung, J, Ladha, A, Jin, X, Gootenberg, JS, Abudayyeh, OO, Zhang, F, Horthongkham, N & Uttamapinant, C 2020, 'Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA', Nature Biomedical Engineering, vol. 4, no. 12, pp. 1140-1149. https://doi.org/10.1038/s41551-020-00603-x

TY - JOUR

T1 - Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA

AU - Patchsung, Maturada

AU - Jantarug, Krittapas

AU - Pattama, Archiraya

AU - Aphicho, Kanokpol

AU - Suraritdechachai, Surased

AU - Meesawat, Piyachat

AU - Sappakhaw, Khomkrit

AU - Leelahakorn, Nattawat

AU - Ruenkam, Theerawat

AU - Wongsatit, Thanakrit

AU - Athipanyasilp, Niracha

AU - Eiamthong, Bhumrapee

AU - Lakkanasirorat, Benya

AU - Phoodokmai, Thitima

AU - Niljianskul, Nootaree

AU - Pakotiprapha, Danaya

AU - Chanarat, Sittinan

AU - Homchan, Aimorn

AU - Tinikul, Ruchanok

AU - Kamutira, Philaiwarong

AU - Phiwkaow, Kochakorn

AU - Soithongcharoen, Sahachat

AU - Kantiwiriyawanitch, Chadaporn

AU - Pongsupasa, Vinutsada

AU - Trisrivirat, Duangthip

AU - Jaroensuk, Juthamas

AU - Wongnate, Thanyaporn

AU - Maenpuen, Somchart

AU - Chaiyen, Pimchai

AU - Kamnerdnakta, Sirichai

AU - Swangsri, Jirawat

AU - Chuthapisith, Suebwong

AU - Sirivatanauksorn, Yongyut

AU - Chaimayo, Chutikarn

AU - Sutthent, Ruengpung

AU - Kantakamalakul, Wannee

AU - Joung, Julia

AU - Ladha, Alim

AU - Jin, Xin

AU - Gootenberg, Jonathan S.

AU - Abudayyeh, Omar O.

AU - Zhang, Feng

AU - Horthongkham, Navin

AU - Uttamapinant, Chayasith

PY - 2020/12

Y1 - 2020/12

N2 - Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)—the virus that causes coronavirus disease 2019 (COVID-19)—in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.

AB - Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)—the virus that causes coronavirus disease 2019 (COVID-19)—in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.

UR - https://www.scopus.com/pages/publications/85089869548

U2 - 10.1038/s41551-020-00603-x

DO - 10.1038/s41551-020-00603-x

M3 - 文章

C2 - 32848209

AN - SCOPUS:85089869548

SN - 2157-846X

VL - 4

SP - 1140

EP - 1149

JO - Nature Biomedical Engineering

JF - Nature Biomedical Engineering

IS - 12

ER -

Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA

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