A CRISPR Switch Integrated with Strand Displacement Amplification for Binary Channel Detection of SARS-CoV-2 Gene Fragments and Infectious Diagnosis

The development of sensitive, accurate, and multimodal approaches for the detection of viral gene fragments and the diagnosis of infections is essential for effective pandemic management across various contexts. This study introduces a CRISPR switch integrated with strand displacement amplification (SDA) for the binary channel detection of SARS-CoV-2 gene fragments and the diagnosis of SARS-CoV-2 infections. In the conventional channel, a specific single gene fragment can directly facilitate the formation of a three-way junction, thereby initiating the SDA process and resulting in the production of a substantial amount of single-stranded DNA. In the logical channel, two gene fragments can first induce the release of a substitute, which subsequently leads to the formation of the three-way junction and the ensuing SDA process. The single-stranded SDA product acts as the target sequence that activates the CRISPR switch, which performs reporter cleavage functions, thereby generating enhanced and detectable fluorescence signals. This method achieves sensitive and selective detection of SARS-CoV-2 gene fragments, with limits of detection (LODs) of 1.0 aM for the ORF1ab gene and 0.9 aM for the N gene in the conventional channel and 3.7 aM for simultaneous detection of both ORF1ab and N in the logical channel. Furthermore, accurate detection of these gene fragments in real samples obtained from patients exhibiting upper respiratory symptoms was successfully conducted, along with the corresponding diagnosis of SARS-CoV-2 infections. Consequently, this method represents a novel binary channel approach for viral gene detection and holds significant promise for clinical diagnosis and potential future epidemic control.