NSCLC ctDNA点突变检测在指导个体化治疗、耐药监测等方面具有重要意义。但现有技术面临以下难点：血液ctDNA含量低，检测灵敏度不能满足临床需求；难以实现对富含GC序列和吉布斯自由能变差值较小点突变的准确检测；血液样本含有大量野生型靶基因，对检测造成很大干扰。针对上述问题，本项目基于不对称PCR和ARMS PCR原理，构建新型不对称ARMS PCR技术，富集并扩增点突变单链靶基因，提高检测灵敏度；研究Bulge loop位置和碱基数对探针特异性的精细调节规律，进而设计高特异性Bulge loop探针；针对野生型靶基因设计信号消除探针，联合熵驱动信号放大策略，建立竞争放大体系，进一步提高检测特异性和灵敏度。最后，利用荧光编码微球和磁珠标记Bulge loop探针，建立悬浮芯片检测方法，实现ctDNA点突变的高通量检测。本项目的实施将为NSCLC靶向药物选择和疗效评价提供新的技术支持。

Analysis of circulating tumor DNA (ctDNA) is of great significance for guiding targeted therapy and monitoring drug resistance in patients with non-small cell lung cancer (NSCLC). However, the existing technologies are challenged by the following issues: the sensitivity of most detection methods can hardly meet clinical requirement due to low abundance of target ctDNA in blood; accurate detection of small point mutations is not available for those that are rich in GC content and low in Gibbs free energy change; target mutations are frequently accompanied with a large number of wild-type counterpart, which would greatly interfere with the detection of targets. To solve the above problems, this project aims to construct a new asymmetric ARMS PCR technique based on the synergistic combination of asymmetric PCR and ARMS PCR. It allows effective enrichment and amplification of single-stranded target gene mutations, which will ultimately enhance the detection sensitivity. The effect of the location and base number of bulge loop on the regulation of probe specificity will be firstly elucidated to guide the design of bulge loop probes with high specificity. A competitive amplification system will be established subsequently with signal elimination probes specific to wild-type genes and entropy-driven signal amplification strategy to further improve the specificity and sensitivity. Finally, a new method to detect ctDNA point mutations in NSCLC with high specificity, sensitivity and high throughput will be developed using fluorescent encoding microspheres and magnetic beads labeled with bulge loop probes. The successful approval of this project will provide technical support for the screening and evaluation of drugs for targeted NSCLC therapy.