Open AccessDigital CRISPR/Cas‐Assisted Assay for Rapid and Sensitive Detection of SARS‐CoV‐2
Author(s) -
Park Joon Soo,
Hsieh Kuangwen,
Chen Liben,
Kaushik Aniruddha,
Trick Alexander Y.,
Wang TzaHuei
Publication year - 2021
Publication title -
advanced science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.388
H-Index - 100
ISSN - 2198-3844
DOI - 10.1002/advs.202003564
Subject(s) - crispr , digital polymerase chain reaction , covid-19 , computational biology , recombinase polymerase amplification , nucleic acid , microfluidics , chemistry , real time polymerase chain reaction , nanotechnology , biology , polymerase chain reaction , genetics , materials science , gene , medicine , infectious disease (medical specialty) , disease , pathology
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 d igitization‐ e nhanced C RISPR/Cas‐assisted o ne‐pot vi rus d etection (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) µL −1 of SARS‐CoV‐2 RNA and 20 GE µL −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.