Digital CRISPR/Cas12b-based platform enabled absolute quantification of viral RNA

Abstract

Early and accurate diagnosis of viruses is critical for control of the pandemic. CRISPR/Cas-based detection of nucleic acid is an emerging technology for molecular diagnostics, and has been applied for virus detection. Though these methods have excellent sensitivity and specificity, most of them were not able to measure the quantity of virus. We here developed a droplet digital reverse transcription loop-mediated isothermal amplification (RT-LAMP) enhanced Cas12b-based RNA detection platform (RECD), for quantitative detection of viral RNA. CRISPR/Cas12b, which is more thermally stable than other family members in CRISPR systems, is combined with digital RT-LAMP. Due to the innate characteristic of digital format detection and CRISPR/Cas system, droplet digital RECD (ddRECD) assay enables absolute quantification of viral RNA, with single-molecule sensitivity. We expect the ddRECD assay will be a powerful tool for molecular diagnostics.

Keywords: CRISPR/Cas12b; Digital isothermal amplification; Quantitative diagnosis; Reverse transcription loop-mediated isothermal amplification.