Sensitive and reliable Kawasaki disease related microRNA analysis via self-priming chain extension cascades

Kawasaki disease (KD) is a leading cause of acquired heart disease in children, requiring timely diagnosis. Circulating microRNAs (miRNAs) are promising non-invasive biomarkers for KD, but their accurate quantification is challenged by low abundance, high sequence homology, and complex biological matrices. Herein, we report a self-priming cascade amplification strategy using a structurally stabilized ternary hybridization probe for sensitive and reliable detection of KD-related miRNAs. The rationally designed probe integrates three oligonucleotides into a robust complex, suppressing nonspecific background signals. Upon target recognition, an orchestrated cascade is activated, including target recycling, polymerase-assisted extension, and nicking endonuclease-mediated exponential amplification, ultimately generating a G-quadruplex-enhanced fluorescence readout. Under optimized conditions, this method achieves a detection limit of 0.82 fM with a dynamic range from 1 fM to 10 nM, and excellent discrimination against mismatched sequences. The assay shows outstanding robustness in human serum, with recovery rates of 96.7-103% and strong correlation with qRT-PCR. Key innovations include the stabilized ternary probe that minimizes background noise and the multi-layered amplification cascade that enhances sensitivity, overcoming the trade-off between stability and efficiency in self-priming systems. This isothermal and robust platform holds great promise for early diagnosis of Kawasaki disease and can be extended to other nucleic acid biomarkers.