Isothermal Target and Signaling Probe Amplification Method, Based on a Combination of an Isothermal Chain Amplification Technique and a Fluorescence Resonance Energy Transfer Cycling Probe Technology
Isothermal Target and Signaling Probe Amplification Method, Based on a Combination of an Isothermal Chain Amplification Technique and a Fluorescence Resonance Energy Transfer Cycling Probe Technology
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DOI：
10.1021/ac100606m
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发表时间：
2010-07-15
影响因子：
7.4
通讯作者：
Park, Hyun Gyu
中科院分区：
文献类型：
--
作者：
Jung, Cheulhee;Chung, Ji Won;Park, Hyun Gyu
描述了一种基于新型伊卡（等温链扩增）和荧光共振能量转移循环探针技术（FRET CPT）相结合的用于核酸定量检测的iTPA（等温靶和信号探针扩增）方法。在新的伊卡方法中，该方法依赖于DNA聚合酶的链置换活性和RNase H的RNA降解活性，在四种特别设计的引物存在下发生两个置换事件。这种现象导致靶DNA的强大扩增。由于扩增仅在四种引物杂交后开始，伊卡方法导致对靶序列的高特异性。作为新伊卡方法的一部分，iTPA是通过结合FRET CPT从单个靶分子产生多个荧光信号来实现的。使用所得到的双重靶标和信号传导探针扩增系统，甚至可以在等温条件下成功地检测和定量靶基因的单拷贝水平。
An iTPA (isothermal target and signaling probe amplification) method for the quantitative detection of nucleic acids, based on a combination of novel ICA (isothermal chain amplification) and fluorescence resonance energy transfer cycling probe technology (FRET CPT), is described. In the new ICA method, which relies on the strand displacement activity of DNA polymerase and the RNA degrading activity of RNase H, two displacement events occur in the presence of four specially designed primers. This phenomenon leads to powerful amplification of target DNA. Since the amplification is initiated only after hybridization of the four primers, the ICA method leads to high specificity for the target sequence. As part of the new ICA method, iTPA is achieved by incorporating FRET CPT to generate multiple fluorescence signals from a single target molecule. Using the resulting dual target and signaling probe amplification system, even a single copy level of a target gene can be successfully detected and quantified under isothermal conditions.