基于芯片不对称PCR的单细胞焦磷酸测序新方法及其在乳腺癌个体化精准给药中的应用研究

Studies show that the circulating tumor cell (CTC) can replace tissue for noninvasive diagnosis of tumor and is of significant clinical value in early diagnosis of tumor, real-time monitoring of disease progression, individual treatment and prognostic evaluation. However, the number of CTC captured from the blood is very few and almost at single-cell level, where conventional methods is insufficient in quantitative analysis. Current single cell analysis requires the fluorescence labeling technique and expensive equipment, with high cost, low sensitivity and poor quantificational performance; and thus its application is limited in clinic. In this study, chemically modified glass chip is adopted for in situ nucleic acid amplification by 1 µL PCR, which has higher amplification efficiency and less reagent consumption than traditional PCR. The asymmetric PCR system using improved linear-after-the-Exponential PCR (imLATE-PCR) can produce single-stranded DNA (ssDNA) for pyrosequencing without tedious procedures of template preparation. Pyrosequencing is highly sensitive with no requirement of fluorescence labeling technique, and can be utilized for single cell nucleic acid sequence analysis and single cell gene expression analysis. This novel method for single-cell nucleic acid analysis is simple, efficient and cost-effective. It is applied in detection of drug-resistant mutations in CTCs before and after targeted drug therapy for breast cancer and prognostic gene expression analysis, providing scientific basis for real-time and noninvasive diagnosis of tumor and individual precision medicine.

循环肿瘤细胞（CTC）可以代替组织实现对肿瘤的无创活检，对肿瘤的早期诊断、实时病程监测、个体化药物治疗和预后评估等具有重要临床价值。但血液中可以捕获到的CTC数目极少，达到单细胞水平，常规方法无法定量分析。现有单细胞分析技术需使用荧光标记、检测成本高、灵敏度低、定量性能差、仪器价格昂贵，目前无法在临床普遍应用。本课题拟采用化学修饰的芯片进行1 µL超微体积PCR对单细胞核酸进行原位扩增，与传统PCR相比扩增效率更高、试剂消耗极少；采用不对称PCR扩增获得单链DNA产物，可直接用作焦磷酸测序模板，避免了复杂的传统测序模板制备过程；采用生物发光焦磷酸测序技术既可测定核酸序列，又可进行基因表达量分析，且无需荧光标记、灵敏度高，从而建立一种简便高效低成本的单细胞核酸分析新方法。并将其应用于乳腺癌靶向用药前后CTC耐药基因突变和预后基因表达量的检测，为肿瘤的实时无创诊断和个体化精准给药提供科学依据。

循环肿瘤细胞（CTC）可以代替组织实现对肿瘤的无创活检，对肿瘤的早期诊断、实时病程监测、个体化药物治疗和预后评估等具有重要临床价值。但血液中可以捕获到的CTC数目极少，达到单细胞水平，常规方法无法定量分析。现有单细胞分析技术需使用荧光标记、检测成本高、灵敏度低、定量性能差、仪器价格昂贵，目前无法在临床普遍应用。本课题采用化学修饰的芯片进行1 µL超微体积PCR对单细胞核酸进行原位扩增，与传统PCR相比扩增效率更高、试剂消耗极少；采用不对称PCR扩增获得单链DNA产物，可直接用作焦磷酸测序模板，避免了复杂的传统测序模板制备过程；采用生物发光焦磷酸测序技术既可测定核酸序列，又可进行基因表达量分析，且无需荧光标记、灵敏度高，从而建立一种简便高效低成本的单细胞核酸分析新方法。并将其应用于乳腺癌靶向用药前后CTC耐药基因突变和预后基因表达量的检测，为肿瘤的实时无创诊断和个体化精准给药提供科学依据。