Detection of miRNAs presents a particular challenge because of their limited size, high sequence homo- logy and greatly various expression level. In this work, an ultrasensitive, label-free and isothermal miRNA detect...Detection of miRNAs presents a particular challenge because of their limited size, high sequence homo- logy and greatly various expression level. In this work, an ultrasensitive, label-free and isothermal miRNA detection was developed based on asymmertric hairpin probe, exonuclease I（Exo I） and SYBR Green I. The method employed asymmetric hairpin probe to perform cycled polymerization and Exo I to reduce background signal. In the presence of the target miRNA, the target triggers probe-mediated cycled polymerization reactions to generate lots of dsDNA products. The dsDNA product effectively prevents itself from being degraded by Exo I and permitted the insertion of more fluorescence dye into it to enlarge the fluorescence signal. In the absence of the target miRNA, there was no probe-mediated polymerization reaction, and the probe was digested by Exo I added, which minimized the intercala- tion of fluorescence dye to reduce the background signal. The combination of the probe-mediated cycled polymeriza- tion with the Exo 1-assisted background reduction allows us to achieve a detection limit of 5× 10^-18 mol/L. Owing to its ultrasensitivity, excellent specificity, convenience and low-cost, this method might hold out great promise in miRNA detection.展开更多
基金Supported by the National Natural Science Foundation of China（No.21275043） and the National Basic Research Program of China（No .2009CB421601）.
文摘Detection of miRNAs presents a particular challenge because of their limited size, high sequence homo- logy and greatly various expression level. In this work, an ultrasensitive, label-free and isothermal miRNA detection was developed based on asymmertric hairpin probe, exonuclease I（Exo I） and SYBR Green I. The method employed asymmetric hairpin probe to perform cycled polymerization and Exo I to reduce background signal. In the presence of the target miRNA, the target triggers probe-mediated cycled polymerization reactions to generate lots of dsDNA products. The dsDNA product effectively prevents itself from being degraded by Exo I and permitted the insertion of more fluorescence dye into it to enlarge the fluorescence signal. In the absence of the target miRNA, there was no probe-mediated polymerization reaction, and the probe was digested by Exo I added, which minimized the intercala- tion of fluorescence dye to reduce the background signal. The combination of the probe-mediated cycled polymeriza- tion with the Exo 1-assisted background reduction allows us to achieve a detection limit of 5× 10^-18 mol/L. Owing to its ultrasensitivity, excellent specificity, convenience and low-cost, this method might hold out great promise in miRNA detection.
