In this technical note, we report an easy-to-produce, reverse-transcription-free, and protein-enzyme-free lateral flow assay for detection of viral RNA fragments by taking SARS-CoV-2 ORF1ab and N as target models. Catalytic hairpin assembly is utilized for dual RNA fragment orthogonal reaction to generate copious amounts of opened hairpin duplexes, which bridge DNA-modified gold nanoparticles and capture strands on the strip to induce coloration. The dual RNA fragments are simultaneously visualized during one time of sample flow, and single-base-mismatched nontarget sequences can be differentiated.
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