A Digital CRISPR-based Method for the Rapid Detection and Absolute Quantification of Viral Nucleic Acids

Wu, X.; Chan, C.; Lee, Y. H.; Springs, S. L.; Lu, T. K.; Yu, H.

Since the outbreak of SARS-CoV-2, quantitative real-time PCR and CRISPR-based methods have been increasingly used for virus detection because of their speed (1 hour) and sensitivity. However, these methods do not allow for the absolute quantification of virus particles, which could reduce the inter-lab variability and accelerate the virus research. Although there is a digital PCR-based method that has been used for absolute virus quantification, its reaction time (4 hours) is too long for widespread application. Here, we report a rapid digital CRISPR method developed for the absolute quantification of SARS-CoV-2 DNA and Epstein-Barr virus DNA in human samples that yields results within 1 hour. We validated this method using synthetic SARS-CoV-2 DNA and Epstein-Barr viral DNA and compared results with those obtained with digital PCR. Digital CRISPR allows absolute quantification of DNA with a dynamic range from 0.6 to 2027 copies/{micro}L (R2 value > 0.98), without cross reactivity on similar virus and human background DNA. Thus, our digital CRISPR can accurately detect and quantify nucleic acid in 1h without thermal cycling, which provides a 4-fold faster alternative to digital PCR-based virus detection.