基于T7核酸外切酶信号循环放大的microRNA比色传感检测新策略

发布时间：2018-05-11 18:24

  本文选题：酶循环扩增法 + T7核酸外切酶　； 参考：《重庆医科大学》2017年硕士论文

【摘要】：MicroRNA(miRNA)是一类长度为18-23个碱基的内源性、非编码RNA,它与真核细胞的基因调控有着重要的关系。研究表明,miRNA参与了疾病发生发展过程中的信号调节,并且miRNA的异常表达与癌症,神经系统疾病及糖尿病等多种疾病有着紧密的联系。作为一种新型的生物标志物,miRNA具有长度短、丰度低及家族同源性高等特性,使得临床上实现高灵敏检测miRNA面临着巨大的挑战。因此,建立高灵敏的miRNA定量分析方法是目前亟需解决的难题。本课题建立了一种基于T7核酸外切酶“信号关闭”的策略,在均相体系中成功实现了对miRNA的可视化检测。在无靶物质miRNA存在时,单链捕获探针无法被T7核酸外切酶剪切,打开含有G4序列的发夹结构探针,吸光度达到最大值;在靶物质miRNA存在时,miRNA与捕获探针杂交形成DNA/RNA双链,利用T7核酸外切酶,剪切双链中的捕获探针,使得捕获探针浓度大大减小,被打开的发夹探针数量减少,从而导致信号降低。在最优的实验条件下,miRNA最低检测限可达到0.6 p M(S/N=3),在10 p M到100 n M的线性范围内,吸光度的与miRNA的浓度呈线性关系。该方法对同源miRNA具有很高的区分度,同时在复杂基质中对miRNA有着良好的分析性能。因此,该传感策略将有可能为miRNA的检测提供了新方法,为临床相关疾病的诊断及治疗提供了技术支持。
[Abstract]:MicroRNAs miRNAs are a class of endogenous, non-coding RNAs with a length of 18-23 bases, which have an important relationship with the gene regulation of eukaryotic cells. Studies have shown that miRNAs are involved in signal regulation in the process of disease development, and the abnormal expression of miRNA is closely related to many diseases, such as cancer, nervous system diseases, diabetes mellitus and so on. As a new biomarker, miRNA has the characteristics of short length, low abundance and high homology. Therefore, it is an urgent problem to establish a sensitive miRNA quantitative analysis method. In this paper, a strategy of signal shutoff based on T7 nucleic acid exonuclease was established, and the visual detection of miRNA was successfully realized in homogeneous system. In the absence of target material miRNA, the single strand capture probe could not be cut by the T7 nucleic acid exonuclease, and the hairpin structure probe containing G4 sequence was opened, and the absorbance reached the maximum. In the presence of the target material miRNA, the miRNA was hybridized with the capture probe to form a double strand of DNA/RNA. By using T7 nucleic acid exonuclease, the capture probe in the double strand is cut down, the concentration of the capture probe is greatly reduced and the number of hairpin probes opened is reduced, which leads to the decrease of the signal. Under the optimal experimental conditions, the minimum detection limit of miRNA can reach 0.6 p MN / N ~ (3 +). In the linear range of 10 pm to 100nM, the absorbance has a linear relationship with the concentration of miRNA. This method has high discrimination for homologous miRNA and good analytical performance for miRNA in complex matrix. Therefore, this sensing strategy may provide a new method for the detection of miRNA and provide technical support for the diagnosis and treatment of clinically-related diseases.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R440

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相关期刊论文 前1条

1 成永强;李正平;王愈聪;范永山;;MicroRNA分析方法进展[J];化学进展;2010年08期

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