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

doi:10.1038/s41551-020-00603-x

. 2020 Dec;4(12):1140-1149.

doi: 10.1038/s41551-020-00603-x. Epub 2020 Aug 26.

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

Maturada Patchsung^#¹, Krittapas Jantarug^#¹, Archiraya Pattama^#², Kanokpol Aphicho^#¹, Surased Suraritdechachai^#¹, Piyachat Meesawat^#¹, Khomkrit Sappakhaw^#¹, Nattawat Leelahakorn¹, Theerawat Ruenkam¹, Thanakrit Wongsatit¹, Niracha Athipanyasilp², Bhumrapee Eiamthong¹, Benya Lakkanasirorat¹, Thitima Phoodokmai¹, Nootaree Niljianskul³, Danaya Pakotiprapha⁴, Sittinan Chanarat⁴, Aimorn Homchan⁴, Ruchanok Tinikul⁴, Philaiwarong Kamutira⁴, Kochakorn Phiwkaow¹, Sahachat Soithongcharoen¹, Chadaporn Kantiwiriyawanitch¹, Vinutsada Pongsupasa¹, Duangthip Trisrivirat¹, Juthamas Jaroensuk¹, Thanyaporn Wongnate¹, Somchart Maenpuen⁵, Pimchai Chaiyen¹, Sirichai Kamnerdnakta⁶, Jirawat Swangsri⁶, Suebwong Chuthapisith⁶, Yongyut Sirivatanauksorn⁶, Chutikarn Chaimayo², Ruengpung Sutthent², Wannee Kantakamalakul², Julia Joung^{7

8

9

10}, Alim Ladha^{7

8

9

10}, Xin Jin^{8

9

11

12}, Jonathan S Gootenberg^{9

13}, Omar O Abudayyeh^{9

13}, Feng Zhang^{7

8

9

10

13

14}, Navin Horthongkham¹⁵, Chayasith Uttamapinant¹⁶

Affiliations

¹ School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, Thailand.
² Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
³ PTT Public Company Limited, Bangkok, Thailand.
⁴ Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok, Thailand.
⁵ Department of Biochemistry, Faculty of Science, Burapha University, Chonburi, Thailand.
⁶ Department of Surgery, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
⁷ Howard Hughes Medical Institute, Cambridge, MA, USA.
⁸ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁹ McGovern Institute for Brain Research at MIT, Cambridge, MA, USA.
¹⁰ Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
¹¹ Society of Fellows, Harvard University, Cambridge, MA, USA.
¹² Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.
¹³ Massachusetts Consortium for Pathogen Readiness, Boston, MA, USA.
¹⁴ Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA.
¹⁵ Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. navin.hor@mahidol.edu.
¹⁶ School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, Thailand. chayasith.u@vistec.ac.th.

^# Contributed equally.

PMID: 32848209
DOI: 10.1038/s41551-020-00603-x

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

Maturada Patchsung et al. Nat Biomed Eng. 2020 Dec.

. 2020 Dec;4(12):1140-1149.

doi: 10.1038/s41551-020-00603-x. Epub 2020 Aug 26.

Authors

Affiliations

¹ School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, Thailand.
² Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
³ PTT Public Company Limited, Bangkok, Thailand.
⁴ Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok, Thailand.
⁵ Department of Biochemistry, Faculty of Science, Burapha University, Chonburi, Thailand.
⁶ Department of Surgery, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
⁷ Howard Hughes Medical Institute, Cambridge, MA, USA.
⁸ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁹ McGovern Institute for Brain Research at MIT, Cambridge, MA, USA.
¹⁰ Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
¹¹ Society of Fellows, Harvard University, Cambridge, MA, USA.
¹² Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.
¹³ Massachusetts Consortium for Pathogen Readiness, Boston, MA, USA.
¹⁴ Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA.
¹⁵ Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. navin.hor@mahidol.edu.
¹⁶ School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, Thailand. chayasith.u@vistec.ac.th.

^# Contributed equally.

PMID: 32848209
DOI: 10.1038/s41551-020-00603-x

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.

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Comment in

Rapid and frequent testing.
[No authors listed] [No authors listed] Nat Biomed Eng. 2020 Dec;4(12):1121-1122. doi: 10.1038/s41551-020-00670-0. Nat Biomed Eng. 2020. PMID: 33293723 No abstract available.

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References

1. Vogels, C. B. F. et al. Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT–qPCR primer–probe sets. Nat. Microbiol. https://doi.org/10.1038/s41564-020-0761-6 (2020).
1. Kelly-Cirino, C. D. et al. Importance of diagnostics in epidemic and pandemic preparedness. BMJ Glob. Health 4, e001179 (2019). - DOI
1. Piepenburg, O., Williams, C. H., Stemple, D. L. & Armes, N. A. DNA detection using recombination proteins. PLoS Biol. 4, e204 (2006). - DOI
1. Notomi, T. et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, e63 (2000). - DOI
1. Rabe, B. A. & Cepko, C. SARS-CoV-2 detection using an isothermal amplification reaction and a rapid, inexpensive protocol for sample inactivation and purification. Preprint at https://doi.org/10.1101/2020.04.23.20076877 (2020).

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[1] Vogels, C. B. F. et al. Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT–qPCR primer–probe sets. Nat. Microbiol. https://doi.org/10.1038/s41564-020-0761-6 (2020).

[2] Vogels, C. B. F. et al. Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT–qPCR primer–probe sets. Nat. Microbiol. https://doi.org/10.1038/s41564-020-0761-6 (2020).

[3] Kelly-Cirino, C. D. et al. Importance of diagnostics in epidemic and pandemic preparedness. BMJ Glob. Health 4, e001179 (2019). - DOI

[4] Kelly-Cirino, C. D. et al. Importance of diagnostics in epidemic and pandemic preparedness. BMJ Glob. Health 4, e001179 (2019). - DOI

[5] Piepenburg, O., Williams, C. H., Stemple, D. L. & Armes, N. A. DNA detection using recombination proteins. PLoS Biol. 4, e204 (2006). - DOI

[6] Piepenburg, O., Williams, C. H., Stemple, D. L. & Armes, N. A. DNA detection using recombination proteins. PLoS Biol. 4, e204 (2006). - DOI

[7] Notomi, T. et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, e63 (2000). - DOI

[8] Notomi, T. et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, e63 (2000). - DOI

[9] Rabe, B. A. & Cepko, C. SARS-CoV-2 detection using an isothermal amplification reaction and a rapid, inexpensive protocol for sample inactivation and purification. Preprint at https://doi.org/10.1101/2020.04.23.20076877 (2020).

[10] Rabe, B. A. & Cepko, C. SARS-CoV-2 detection using an isothermal amplification reaction and a rapid, inexpensive protocol for sample inactivation and purification. Preprint at https://doi.org/10.1101/2020.04.23.20076877 (2020).

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

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

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