基于CRISPR/SpRY等温扩增和Au@PdUss生物传感器的肺癌ctDNA精准检测新方法

循环肿瘤DNA(ctDNA)检测作为一种非侵袭性“液体活检”手段，可为肺癌的早期诊断、诊疗预后和个体化治疗等提供重要信息，具有极高的研究价值和临床意义。但是，ctDNA在血液中含量稀少，而且常受到野生型高背景的干扰，现有技术难于实现低丰度ctDNA的精准测定。为此，本项目拟构建一种基于CRISPR/SpRY等温扩增和Au@Pd生物传感器的肺癌ctDNA精准检测新方法。将完成如下工作：①构建基于海胆状Au@Pd纳米粒子的生物传感器，实现ctDNA的超敏快速检测；②利用升级改造版“基因魔剪”CRISPR/SpRY和等温指数扩增反应创新联用，SpRY实现单碱基突变点也能精准识别，从而有效提升检测的特异性；③探索本项目方法在肺癌ctDNA-EGFR突变检测中的应用，为肺癌ctDNA早期诊断提供依据和技术支撑。本项目方法可实现血液中ctDNA精准特异的测定，为“液体活检”中分子诊断提供新思路。

As a kind of non-invasive detection of "liquid biopsy", circulating tumor DNA (ctDNA) play an important role in early diagnosis, prognosis and individualized treatment of tumor diseases, with high research value and clinical significance. However, ctDNA are rare in the peripheral blood, and often interfered by the high background of wild type, current ctDNA detection methods are difficult to achieve accurate detection. In order to achieve accurate detection of ctDNA, we propose a novel method based on CRISPR/SpRY isothermal amplification and Au@Pd biosensor. We plan to accomplish the following tasks: ①Construction of a biosensor based on Au@Pd urchin-shaped nanostructures to realize the sensitive and fast detection of ctDNA; ②Innovative combination of the newly modified “gene magic scissors” CRISPR/SpRY and isothermal exponential amplification, and even the single-base mutation point could be accurately identified, effectively improve the specificity; ③To explore the application of our method in the detection of EGFR mutation, providing the basis and technical support for ctDNA diagnosis of lung cancer. This study is expected to realize accurate and specific detection of ctDNA, and provide a new idea for molecular diagnosis in "liquid biopsy".