基于多重乳滴数字PCR芯片高灵敏检测肺癌循环DNA中KRAS基因突变的研究

Resistance presents a major problem to lung cancer molecular targeted therapy. KRAS gene mutation is one of the mechanisms associated with resistance to EGFR-TKIs. So, the accurate and efficient determination of KRAS gene mutations is very important for patients with targeted drug therapy. Currently, targeted drug therapy for lung cancer is based on the detection of mutant DNA in tumor tissues, but tumor samples are sometimes difficult to obtain. Circulating tumor DNA (ctDNA) in peripheral blood is a "liquid biopsy" that contains tumor informations including gene mutations, but it has brought great challenges to the detection methods due to its low content. Based on the detection of single nucleic acid biomarker with a droplet digital PCR chip in our previous research, this project will integrate microfluidic chip with droplet digital PCR techniques, by designing four reaction zones on the chip and combining the site-specific TaqMan-MGB dual-labeled fluorescent probes, to establish a highly sensitive and specific method for simultaneous detection of multiple KRAS gene mutations in ctDNA, and we will further study the consistency of KRAS gene mutations in peripheral blood and tumor tissue of lung cancer patients. This project will provide technology for the detection of rare mutant DNA, and the basis for the targeted drug therapy of lung cancer, which has great application value and social significance.

肺癌靶向药物治疗的一个重要问题就是耐药。KRAS基因突变是患者对EGFR-TKIs产生耐药的机制之一。准确、高效地测定KRAS基因突变，对患者靶向药物治疗具有重要意义。目前肺癌靶向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。外周血循环肿瘤DNA（ctDNA）的“液体活检”可反映肿瘤基因突变信息，但因其含量低而给检测方法带来了巨大的挑战。本项目在前期乳滴数字PCR芯片检测单一核酸标志物的研究基础上，拟将微流控芯片和乳滴数字PCR技术结合，通过在芯片上设计四个反应区，结合位点特异性TaqMan-MGB双标记荧光探针，建立外周血ctDNA中KRAS基因多种突变高灵敏、高特异的检测方法，实现对多个靶标的同时检测，并进一步研究外周血与组织样本中KRAS基因突变的一致性。本项目的研究成果将为微量突变DNA的检测提供技术，为肺癌靶向药物治疗提供依据，具有重要的应用价值和社会意义。

肺癌靶向药物治疗的一个重要问题就是耐药。KRAS基因突变是患者对EGFR-TKIs产生耐药的机制之一。准确、高效地测定KRAS基因突变，对患者靶向药物治疗具有重要意义。外周血中ctDNA可反映肿瘤基因突变信息，并且在早期和晚期肿瘤患者中都可以检测到。由于外周血ctDNA中KRAS突变体非常少，而数字PCR可以在大量野生型DNA背景中鉴定出微量突变体，与测序法及QPCR法相比，突变基因的检测灵敏度可以提高1000倍。本项目的主要研究内容如下：（1）构建了7种突变型KRAS基因质粒和野生型KRAS基因质粒，并优化反应中的引物、探针浓度及探针的特异性，建立了QPCR体系用于KRAS基因突变的检测，并研究了所构建的QPCR体系对KRAS基因突变检测的灵敏度和特异性，结果表明QPCR只能检测到1%-5%的不同类型KRAS基因突变，与文献报道的一致；（2）与重庆大学光电工程学院微系统研究中心合作，设计了单一体系和多重引物预存储的微腔阵列式数字PCR芯片，考察了多重引物预存储数字PCR芯片的交叉污染现象，并将芯片用于KRAS基因突变的检测。本项目所研制的芯片可实现样品的快速、自动的数字化分解，同时也成功实现了芯片上多重引物的分区预存储技术方案，有望为肿瘤的精准医疗提供一种有效的分子生物学检测手段。. 在项目的研究基础上，结合依托单位的条件，我们构建了一种基于锁核酸探针结合杂交链反应的电化学生物传感器用于KRAS G12D基因突变的检测，检测线性范围为1×10-15 ~ 1×10-10 mol/L，检测限为0.29 fM。