An amplification-free digital droplet assay for influenza A viral RNA based on CRISPR/Cas13a

Most of the CRISPR-based RNA detection methods are combined with amplification to improve sensitivity, which lead to some drawbacks such as aerosol pollution, complicated operation, and amplification bias. To address the above issues, we developed a digital detection method for influenza A viral RNA based on droplet microfluidics and CRISPR/Cas13a without polymerase chain reaction. We used a microsphere coupled to a capture probe to extract and concentrate the target RNA from the samples, and then restricted the target-induced CRISPR/Cas13a cleavage event to microfluidic droplets, thus enhancing the local signal intensity and enabling single-molecule detection. With a detection limit of 10 copies per μL, influenza A viral RNA can be detected in less than 1 h. Both clinical and synthetic series samples were used to validate the assay's performance. With the help of this direct RNA diagnostic method, a variety of RNA molecules can be easily and accurately detected at the single-molecule level. This research has broad prospects in clinical applications.