非小细胞肺癌(NSCLC) ctDNA的检测对于肿瘤的分期和靶向治疗具有重要意义。然而，ctDNA含量极低且伴有大量正常细胞来源的优势野生型游离DNA。因此，NSCLC ctDNA的检测需要解决以下问题：优势野生型游离DNA对ctDNA的干扰；ctDNA突变位点的准确阅读。为了解决上述难题：针对优势游离DNA的干扰，我们利用链置换引物介导的PCR实现野生型游离DNA的背景压制和稀有ctDNA的有效富集；针对突变位点的准确阅读，采用分子信标介导的熔解曲线并建立合适的温度判读规则来实现突变位点的准确检测，并实现稀有突变的相对定量分析。探索链置换引物介导的多重PCR体系的复杂程度实现多个靶基因的单反应体系同时扩增，并结合分子信标介导的熔解曲线实现突变位点的准确阅读。该项目的实施将为NSCLC稀有变异检测和临床应用提供全新技术平台，为肿瘤早期诊断提供依据，为肿瘤分期、转移和靶向治疗提供信息保障。

Detection of non-small cell lung cancer (NSCLC) ctDNA is important for tumor staging and targeted therapy. However, ctDNA content is extremely low and accompanied by a large number of dominant wild-type free DNA from normal cell. Therefore, the detection of NSCLC ctDNA needs to solve the following scientific problems: the interference of dominant wild-type free DNA to rare mutant ctDNA; accurate detection of ctDNA mutation sites. In order to solve the above problems: for the interference of dominant free DNA, we use strand displacement primer-mediated PCR to achieve background suppression of wild-type free DNA and efficient enrichment of rare ctDNA; for accurate mutation sites detection, molecular beacons mediated high-sesolution melting curve analysis are used and appropriate temperature interpretation rules are established to achieve accurate detection of the mutation sites, at the same time relative quantitative analysis of rare mutations are achieved. Exploring the the multiplex PCR-mediated multiplex PCR to reduce the complexity of the single-reaction system of multiple target genes, by the combination of molecular beacon-mediated melting curve to achieve accurate reading of the mutation site. The implementation of the project will provide a new technology platform for NSCLC rare mutation detection and clinical application, provide a basis for early diagnosis of tumors, and provide information guarantee for tumor staging, metastasis and targeted therapy.