Abstract
Digital PCR (dPCR) is the most sensitive method for virus detection by accurate quantifying the nucleic acids. However, the requirement of sophisticated and expensive instrumentations greatly limits its application in clinical diagnosis particularly in poorly resourced settings. Herein, we developed a simple digital assay based on polydisperse droplet digital recombinase-aided amplification/reverse transcription recombinase-aided amplification dependent CRISPR/Cas12a system (PddRaaCas/PddRRaaCas) for viral DNA/RNA detection with minimal instruments. This assay does not require any microfluidic chip for droplet generation and can be completed about 1 hour. We found that the PddRaaCas and PddRRaaCas enabled to detect 0.015 fM target DNA and 10 fM target RNA respectively. In addition, we applied this method to detect human papillomavirus type 18 (HPV18) and severe acute respiratory syndrome coronavirus (SARS-CoV-2) in clinical samples, which represented the detection of DNA virus and RNA virus respectively. The sensitivity and specificity were 100 % and 100 % for both DNA and RNA virus detection respectively. Taken together, these results highlight that PddRaaCas/PddRRaaCas is a sensitive, rapid, and easy-to-use strategy for the detection of DNA/RNA viruses, which has great potential application for the control of infectious diseases.
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