文献类型：期刊文献

英文题名：One-Step, Multiplexed Fluorescence Detection of microRNAs Based on Duplex-Specific Nuclease Signal Amplification

作者：Yin, Bin-Cheng[1];Liu, Yu-Qiang[1];Ye, Bang-Ce[1]

机构：[1]E China Univ Sci & Technol, Lab Biosyst & Microanal, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2012

卷号：134

期号：11

起止页码：5064

外文期刊名：JOURNAL OF THE AMERICAN CHEMICAL SOCIETY

收录：;EI(收录号：20121314897236);WOS:【SCI-EXPANDED(收录号:WOS:000302191900016)】；

基金：Supported by China NSF21075040, NSF31101285, and the Shanghai fund (11XD1401900, 11nm0502500). We are grateful to Prof. Hai-Bo Cai and Dr. Wei-Wei Zhang for kindly assisting in the cell-culture, and the supporting from Hitachi, Ltd.

语种：英文

外文关键词：Diagnosis - RNA - Clinical research - Fluorescence - Amplification

摘要：Traditional molecular beacons, widely applied for detection of nucleic acids, have an intrinsic limitation on sensitivity, as one target molecule converts only one beacon molecule to its fluorescent form. Herein, we take advantage of the duplex-specific nuclease (DSN) to create a new signal-amplifying mechanism, duplex-specific nuclease signal amplification (DSNSA), to increase the detection sensitivity of molecular beacons (Taqman probes). DSN nuclease is employed to recycle the process of target-assisted digestion of Taqman probes, thus, resulting in a significant fluorescence signal amplification through which one target molecule cleaves thousands of probe molecules. We further demonstrate the efficiency of this DSNSA strategy for rapid direct quantification of multiple miRNAs in biological samples. Our experimental results showed a quantitative measurement of sequence-specific miRNAs with the detection limit in the femtomolar range, nearly 5 orders of magnitude lower than that of conventional molecular beacons. This amplification strategy also demonstrated a high selectivity for discriminating differences between miRNA family members. Considering the superior sensitivity and specificity, as well as the multiplex and simple-to-implement features, this method promises a great potential of becoming a routine tool for simultaneously quantitative analysis of multiple miRNAs in tissues or cells, and supplies valuable information for biomedical research and clinical early diagnosis.