Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA.
Maturada PatchsungKrittapas JantarugArchiraya PattamaKanokpol AphichoSurased SuraritdechachaiPiyachat MeesawatKhomkrit SappakhawNattawat LeelahakornTheerawat RuenkamThanakrit WongsatitNiracha AthipanyasilpBhumrapee EiamthongBenya LakkanasiroratThitima PhoodokmaiNootaree NiljianskulDanaya PakotipraphaSittinan ChanaratAimorn HomchanRuchanok TinikulPhilaiwarong KamutiraKochakorn PhiwkaowSahachat SoithongcharoenChadaporn KantiwiriyawanitchVinutsada PongsupasaDuangthip TrisriviratJuthamas JaroensukThanyaporn WongnateSomchart MaenpuenPimchai ChaiyenSirichai KamnerdnaktaJirawat SwangsriSuebwong ChuthapisithYongyut SirivatanauksornChutikarn ChaimayoRuengpung SutthentWannee KantakamalakulJulia JoungAlim LadhaXin JinJonathan S GootenbergOmar O AbudayyehFeng ZhangNavin HorthongkhamChayasith UttamapinantPublished in: Nature biomedical engineering (2020)
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.
Keyphrases
- sars cov
- respiratory syndrome coronavirus
- coronavirus disease
- real time pcr
- nucleic acid
- loop mediated isothermal amplification
- label free
- crispr cas
- patients undergoing
- high throughput
- healthcare
- emergency department
- risk assessment
- coronary artery disease
- transcription factor
- gene expression
- drinking water
- atrial fibrillation
- nitric oxide
- single molecule
- hydrogen peroxide
- percutaneous coronary intervention
- dna methylation