A CRISPR/Cas13a system based on a dumbbell-shaped hairpin combined with DNA-PAINT to establish the DCP-platform for highly sensitive detection of Hantaan virus RNA.

Rapid and sensitive detection of specific RNA sequences is crucial for clinical diagnosis, surveillance, and biotechnology applications. Currently, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard for RNA detection; however, it is associated with long processing time, complex procedures, and a high false-positive rate. To address these challenges, we developed a novel sensing platform based on CRISPR/Cas13a that incorporates a dumbbell-shaped hairpin and DNA-PAINT for rapid, highly specific, and sensitive RNA analysis. By leveraging the CRISPR/Cas13a system, this platform enables the cleavage of dumbbell-shaped hairpins, which subsequently allows the cleaved primers to initiate cyclic amplification of fluorescent signals. These signals are further enhanced by the binding and dissociation phenomena inherent to DNA-PAINT technology, ultimately achieving remarkable triple signal amplification. Additionally, the system effectively discriminates Hantaan virus RNA from Seoul virus in real samples. Importantly, the platform can be easily adapted for the detection of other RNAs by simply reconfiguring the hybridization region of crRNA. In conclusion, this platform represents a "five-in-one" RNA detection approach that integrates reliability, versatility, robustness, high specificity, and superior quantitative capabilities. It provides novel insights for direct RNA detection based on CRISPR/Cas13a and demonstrates significant potential for advancement in viral diagnostics.