Sensitive and Reliable Kawasaki Disease Related MicroRNA Analysis via Self-Priming Chain Extension Cascades

Abstract

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.