Multiplex detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction

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• 作者：Yacui Liu ; Jiangyan Zhang ; Jingxiao Tian…
• 关键词：Multiplex microRNA detection ; T7 exonuclease ; Nuclease ; assisted cyclic amplification ; Molecular beacon
• 刊名：Analytical and Bioanalytical Chemistry
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：409
• 期：1
• 页码：107-114
• 全文大小：
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Analytical Chemistry; Biochemistry, general; Laboratory Medicine; Characterization and Evaluation of Materials; Food Science; Monitoring/Environmental Analysis;
• 出版者：Springer Berlin Heidelberg
• ISSN：1618-2650
• 卷排序：409

文摘

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.