期刊
ADVANCED SCIENCE
卷 8, 期 5, 页码 -出版社
WILEY
DOI: 10.1002/advs.202003564
关键词
CRISPR/Cas; diagnostics; digital; nucleic acid; SARS-CoV-2
资金
- National Institutes of Health [R01AI138978, R01AI137272]
- Sherrilyn and Ken Fisher Center for Environmental Infectious Diseases at Johns Hopkins University [FCDP-010ZHA2020]
The digital-enhanced CRISPR/Cas-assisted one-pot virus detection (deCOViD) is a novel assay that allows qualitative and quantitative detection of SARS-CoV-2 in a very short time with high sensitivity and a broad dynamic range. It outperforms current benchtop-based counterparts and represents one of the fastest and most sensitive CRISPR/Cas-assisted SARS-CoV-2 detection methods to date.
The unprecedented demand for rapid diagnostics in response to the COVID-19 pandemic has brought the spotlight onto clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)-assisted nucleic acid detection assays. Already benefitting from an elegant detection mechanism, fast assay time, and low reaction temperature, these assays can be further advanced via integration with powerful, digital-based detection. Thus motivated, the first digital CRISPR/Cas-assisted assay-coined digitization-enhanced CRISPR/Cas-assisted one-pot virus detection (deCOViD)-is developed and applied toward SARS-CoV-2 detection. deCOViD is realized through tuning and discretizing a one-step, fluorescence-based, CRISPR/Cas12a-assisted reverse transcription recombinase polymerase amplification assay into sub-nanoliter reaction wells within commercially available microfluidic digital chips. The uniformly elevated digital concentrations enable deCOViD to achieve qualitative detection in <15 min and quantitative detection in 30 min with high signal-to-background ratio, broad dynamic range, and high sensitivity-down to 1 genome equivalent (GE) mu L-1 of SARS-CoV-2 RNA and 20 GE mu L-1 of heat-inactivated SARS-CoV-2, which outstrips its benchtop-based counterpart and represents one of the fastest and most sensitive CRISPR/Cas-assisted SARS-CoV-2 detection to date. Moreover, deCOViD can detect RNA extracts from clinical samples. Taken together, deCOViD opens a new avenue for advancing CRISPR/Cas-assisted assays and combating the COVID-19 pandemic and beyond.
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