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

• Main Authors: 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
• Other Authors: PTT Public Company Limited
• Format: Article
• Published: 2020
• Subjects: Biochemistry, Genetics and Molecular Biology; Chemical Engineering; Computer Science; Engineering; Medicine
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/59008
• Institution: Mahidol University

© 2020, The Author(s), under exclusive licence to Springer Nature Limited. 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.