Molecular aptamer beacon-based dual-mode exponential isothermal amplification for microRNA detection

Due to the limiting factors associated with microRNAs (miRNAs), including their low abundance, short sequence length, and high family sequence homology, the gold standard for miRNA detection does not meet the requirements for rapid and low-cost clinical detection of miRNAs in biological samples. To this end, we developed a one-pot method called molecular aptamer beacon-utilized exponential isothermal amplification (MABEXIA) for analyzing and detecting miRNAs. This novel devolved method was analyzed using both a fluorescent dye and a horseradish peroxidase (HRP) mediated chemiluminescence reaction with luminol and hydrogen peroxide for miRNA detection. The principle of the fluorescence platform involves hybridization of the target miRNA with MAB, which results in the formation of a large amount of double-stranded (ds) DNA products under the dual catalytic action of a DNA polymerase (Klenow Fragment) and nicking endonuclease (Nb.BbvCI) at a constant temperature. Moreover, this method exhibited good detection performance and was successfully applied to 1% human serum for miRNA detection. In contrast to the fluorescence method, the chemiluminescence approach achieved miRNA detection through the targeted interaction between the activated streptavidin (SA) aptamer within the dsDNA product and SA-coated magnetic beads. This method showed a good linear relationship in the ranges of 50 fM to 50 pM for miRNA detection. Finally, this chemiluminescence approach has a detection ability comparable to that of the gold standard for miRNA detection.