[One-pot isothermal index amplification combined with molecular beacons for ultrasensitive miRNA analysis]

A hairpin-molecular-beacon co-fuelled, single-tube isothermal exponential amplification methodology (HM-EXPAR) was established to enable the rapid and ultrasensitive quantification of miRNA. From January to August 2025, the Department of Laboratory Medicine of People's Hospital of Pidu District, Chengdu City carried out a novel miRNA detection technology study. Taking miR-122 as the model target, a 3'-C3-spacer-capped hairpin probe (HP) and a molecular beacon (MB) were rationally designed; the secondary structures were optimized using NUPACK (Nucleic Acid Package) to evaluate the feasibility of HM-EXPAR. The cycle number corresponding to the maximum slope on the real-time fluorescence curve (point of inflection, POI) is used as the quantitative parameter, the concentrations of HP, MB, Vent(exo-) DNA polymerase and Nt.BstNBI nicking endonuclease were systematically titrated, and the assay was subsequently benchmarked for sensitivity, single-base specificity, intra-and inter-assay precision, versatility, spike-recovery accuracy, and concordance with gold-standard RT-qPCR.The results showed that NUPACK analysis confirmed that the free energy of the HP/miR-122 and MB/Trigger duplexes decreased significantly, indicating excellent thermodynamic stability and specificity, thus verifying the feasibility of the method. The established HM-EXPAR assay displayed excellent linearity over two concentration ranges (100 nmol/L-10 pmol/L and 10 pmol/L-10 fmol/L) under the optimized conditions of 55 ℃ for 30 min (R²≥0.996), with a limit of detection as low as 0.17 fmol/L and a coefficient of variation below 3%. The response ratio for a single-base mismatch was 0.65, while those for other homologous miRNAs were≤0.20, indicating strong anti-interference capability. It also exhibits versatility for the detection of multiple miRNA targets. Spike-recovery experiments in 5% human serum yielded recovery rates of 101.4%-112.0%, and the results showed good consistency with those obtained by RT-qPCR. In conclusion,the HM-EXPAR strategy developed herein enables the ultrasensitive quantification of miRNA within 30 min, covers an 8-log linear dynamic range, and discriminates single-nucleotide variants, providing a robust and user-friendly tool for rapid and high-performance miRNA analysis.