基于核酸侵入信号扩增反应的循环肿瘤DNA突变检测新方法及在临床合理用药中的应用评价

Currently targeted therapy for lung cancer is based on the detection of mutant DNA in tumor tissues, however tumor samples are sometimes difficult to obtain.It has been reported that when a tumor develops into a certain stage, mutant DNA can be detected in the patient's plasma, hence plasma can be an alternative to tumor tissues in which mutant DNA can be detected. However, the fact that detection results may be inconsistent between plasma DNA and tissue DNA by existing methods, limits the clinical application of detection methods for mutant DNA in plasma. Our group have studied the methodology detecting mutant DNA with an incredible background of other DNA and established an isothermal nucleic acid signal amplification method (Cascade Enzymatic Signal Amplification,CESA), making it possible to detect 0.1% mutant DNA.However, this method is still insufficient for detecting mutant DNA in plasma considering its sensitivity, so a new method is preferred.Taking non-small cell lung cancer as an example, we will establish a new method combining highly sensitive nucleic acid amplification with CESA in order to detect tens of copies of circulating tumor DNA in plasma. Moreover, the gold nanoparticle probe technology will be introduced into this method to achieve a highly specific, highly sensitive, low-cost detection of circulating tumor DNA. By exploring the correlation of plasma tumor DNA and tumor tissue DNA, we will estalish a novel tumor-targeted medication platform oriented by plasma tumor DNA, thus providing a new approach for personalized treatment.

目前肺癌靶向药物治疗是基于肿瘤组织突变DNA的检测结果，但临床肿瘤样本有时难以获得。肿瘤发生发展到一定阶段就可在血浆中检测到突变DNA，因此可用血浆代替肿瘤组织。然而现有检测方法有时会使血浆与肿瘤的检测结果有差异，这限制了血浆突变检测方法的临床应用。本课题组针对高背景下检测微量突变DNA进行了方法学研究，创建了可检测0.1%突变DNA的等温核酸信号扩增法（CESA），然而该方法仍不足以检测血浆中极微量突变DNA，为了建立血浆循环肿瘤DNA突变检测方法，本课题拟以非小细胞肺癌为例,将高灵敏核酸扩增与CESA相耦合，建立高特异性的检测血浆循环肿瘤DNA中数十拷贝的突变DNA新方法，通过引入可视化纳米金检测新技术，实现高特异、高灵敏、低成本的循环肿瘤DNA突变体的检测。通过探讨血浆循环肿瘤DNA与肿瘤组织的相关性，建立基于血浆循环肿瘤DNA指导的肿瘤靶向用药新平台，为临床个体化治疗提供新途径。

.肿瘤组织中的基因突变与血浆中的基因突变存在相关性，因此，能够通过检测血浆中的基因突变情况来监视肿瘤组织中的基因突变，从而指导肿瘤的个体化治疗。然而，由于样本中存在大量仅存在单碱基差异的野生型模板，使得临床突变DNA检测难度很大。本课题首先考察了循环肿瘤DNA的提取方法，建立了能够高效获取循环肿瘤DNA的提取方法；其次，通过优化反应条件与反应程序，建立了耦合扩增反应、核酸侵入反应及纳米金探针杂交反应的高灵敏高特异循环肿瘤DNA突变体检测方法，对灵敏度和特异性的考察结果显示，能够检测低至数十拷贝的核酸分子和混合比例低至0.1%的突变模板，对9例实际样本进行检测，检出2例突变样本，与焦磷酸测序结果一致。以上结果表明，本课题建立的稀有突变体检测方法能够很好的检出肿瘤样本DNA突变情况，这为临床肿瘤DNA突变检测提供了新的手段。