H1N1 Viral RNA Detection via FEN1-assisted Isothermal Amplification based on a Three-Way Junction

초록

Accurate and timely detection of Influenza A virus (H1N1) is essential for effective public health management. Traditional PCR-based methods, though effective, are limited by the need for thermal cycling and are impractical in point-of-care settings. To overcome this, we developed a novel isothermal detection strategy that utilizes the structure-specific activity of flap endonuclease 1 (FEN1) enzyme and the high signal-to-background contrast of a light-up RNA aptamer. In our system, the presence of H1N1 target RNA initiates the formation of a three-way junction (3WJ) structure, subsequently recognized and cleaved by FEN1. The resulting cleavage products then act as primers for a cascade of amplification reactions, leading to the transcription of light-up RNA aptamers. This label-free and amplification-driven method enables rapid and sensitive H1N1 detection with a low limit of detection (LOD) of 516.84 fM. Analysis based on three technical replicates (n = 3) demonstrated high reproducibility with coefficients of variation (CVs) consistently below 10%. Additionally, validation in human serum confirmed reliable recovery rates ranging from 96.52% to 100.22%. Furthermore, the system demonstrates high specificity against various non-target viral RNAs and a wide dynamic range, offering a viable solution for rapid and reliable diagnostics in resource-limited environments.

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