Sensitive and rapid detection of microRNAs using hairpin probes-mediated exponential isothermal amplification

Abstract

MicroRNAs (miRNAs) play significant roles in a diverse range of biological progress and have been regarded as biomarkers and therapeutic targets in cancer treatment. Here, we develop a rapid and sensitive miRNA assay on the basis of exponential isothermal amplification in combination with the hairpin probes, which was designed for sensitive detection of miRNA. The binding of target miRNA with a linear DNA template initiates exponential isothermal amplification reaction (EXPAR) and generates the universal triggers which are complementary to the 3′ protruding end of hairpin probe1(HP1). These triggers function not only as the primers to unfold the hairpin probes through catalysed hairpin assembly(CHA), generating distinct fluorescence signals, but also as the primer to initiate the next EXPAR. Moreover, CHA can release new triggers to initiate EXPAR or CHA. Thus this hairpin probes-mediated exponential isothermal amplification assay exhibits high sensitivity with a detection limit of 3.0×10−15M. More importantly, the isothermal condition and simple fluorescence measurement would greatly promote the development of a fast, point-of-care detection system. It can be completed in an hour which can effectively avoid miRNA from degradation. This hairpin probe-based circular exponential amplification assay holds a great promise for further application in biomedical research and early clinical diagnosis.