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

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 molecule out of wild-type molecule, realizing the analysis of minority somatic mutations in clinical tumor samples, thus providing a novel tool for detecting biomarkers related to personalized medication.

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。

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。本研究通过建立一种结合高灵敏等温扩增、高特异核酸侵入反应和纳米金显色反应的新方法，实现了对体细胞突变的单管检测。首先，我们优化了等温扩增和核酸侵入反应体系的兼容性，对反应缓冲液中各种组分的浓度进行考察，满足了单管反应的必要条件。其次，我们建立了高灵敏的等温扩增反应体系，可实现2.5aM突变分子的检测。再次，我们建立了可耐热耐吸附的纳米金制备方法，同时优化了纳米金杂交反应体系，使阳性和阴性结果得以明确区分。最后，我们建立了高特异性的核酸侵入反应，可对0.1%的体细胞突变体进行检测，实现了高野生型背景下的目标识别。本研究建立的方法操作简单、价格低廉，为临床提供了一种个体化用药相关标志物检测的新工具。