纳米金可视化检测极低丰度体细胞突变的新方法评价及临床应用

项目名称： 纳米金可视化检测极低丰度体细胞突变的新方法评价及临床应用

项目编号： No.31300704

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 生物科学

项目作者： 梁超

作者单位： 中国人民解放军南京军区南京总医院

项目金额： 25万元

中文摘要： 体细胞基因突变是一种重要的个体化用药相关生物标志物。但由于临床样本中含有大量的未突变DNA背景，常规方法难以对其中微量的突变基因进行检测。现有基因突变检测方法存在灵敏度低、检测成本高、仪器设备昂贵等缺点，很难在临床中推广应用。前期工作中，我们已建立能检测低至10个拷贝DNA分子的高灵敏环介导等温核酸扩增方法和能检测0.1%突变的高特异核酸侵入反应。本课题组拟将两者结合起来，从大量野生模板背景中识别突变位点产生大量与突变体相对应的核酸片段，再将该通用的核酸片段转化为能使纳米金探针聚集显色的信号分子。通过肉眼观察纳米金溶液颜色的变化判定样本中是否含有基因突变体，据此建立一种高灵敏高特异、低成本的微量体细胞突变体可视化检测新方法。课题目标是使建立的方法能检测到极微量样本中0.1%以下的基因突变，实现肿瘤样本极微量极低丰度的基因突变体检测，为临床提供一种个体化用药相关标志物检测新工具。

中文关键词： 环介导等温扩增；核酸侵入反应；纳米金；可视化检测；体细胞突变

英文摘要： Somatic mutation are of important biomarkers for personalized medication. However, such mutations coexist with incredible other DNA background in clinical samples, making it a challenge for detection by common methods. Current methods for minority mutation detection have the shortbacks such as low sensitivity, high cost and requirement of expensive instruments, limiting their wide application in clinic. In previous work, we have proposed the Loop-mediated Isothermal Amplification which could detect ten copies of DNA molecules in one reaction and an invasive reaction which could detect 0.1% mutant molecule out of wild-type molucles . In this study, based on Loop-mediated Isothermal Amplification coupled with gold nanoparticle probe assembling, a novel method for visualized detection of minority somatic mutations is proposed, featuring high sensitivity, high specificity and low cost.By our proposed method, templates are amplified by isothermal amplification, then mutant DNA will be discerned from vast background by invasive signal amplification, generating a large amount of corresponding signal molecules, thus signal molecules can cause the aggregation of gold nanoparticles probes. If mutant targets exsit in a specimen, the reaction buffer would turn to red. Our goal is to achieve the detection of 0.1% mutant mol

英文关键词： loop-mediated isothermal amplification；invader reaction；gold nanopartical；visualized；somatic mutation