期刊
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
卷 409, 期 1, 页码 107-114出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-016-0027-6
关键词
Multiplex microRNA detection; T7 exonuclease; Nuclease-assisted cyclic amplification; Molecular beacon
资金
- National Natural Science Foundation of China [21475031, 21605034]
- National Science Foundation of Hebei Province [B2016201031]
- Science Technology Research and Development Guidance Programme Project of Baoding City [15ZF071]
A simple, highly sensitive, and specific assay was developed for the homogeneous and multiplex detection of microRNAs (miRNAs) by combining molecular beacon (MB) probes and T7 exonuclease-assisted cyclic amplification. An MB probe with five base pairs in the stem region without special modification can effectively prevent the digestion by T7 exonuclease. Only in the presence of target miRNA is the MB probe hybridized with the target miRNA, and then digested by T7 exonuclease in the 5' to 3' direction. At the same time, the target miRNA is released and subsequently initiates the nuclease-assisted cyclic digestion process, generating enhanced fluorescence signal significantly. The results show that the combination of T7 exonuclease-assisted cyclic amplification reaction and MB probe possesses higher sensitivity for miRNA detection. Moreover, multiplex detection of miRNAs was successfully achieved by designing two MB probes labeled with FAM and Cy3, respectively. As a result, the method opens a new pathway for the sensitive and multiplex detection of miRNAs as well as clinical diagnosis.
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