用于循环肿瘤DNA检测的数字化信号放大新方法及临床应用研究

项目名称： 用于循环肿瘤DNA检测的数字化信号放大新方法及临床应用研究

项目编号： No.21475151

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 数理科学和化学

项目作者： 周国华

作者单位： 中国人民解放军东部战区总医院

项目金额： 85万元

中文摘要： 肿瘤组织活体来源有限，而循环肿瘤DNA（ctDNA）可在治疗过程中反复取样，对肿瘤的个体化诊疗具有重要意义。但血浆中ctDNA含量极低，难以检测。数字化PCR因其定量精确，已成功用于ctDNA的检测。然而该法使用的荧光探针不通用、特异性也不高；更重要的是，需要采用芯片或流式细胞仪进行开管检测，极易造成假阳性结果。本课题提出将核酸信号放大反应与数字化检测相结合对单分子计数检测的新思路。首先采用结构特异性核酸内切酶反应，将待测靶标放大成核酸信号分子，再采用FRET发卡探针进一步放大，产生放大的荧光信号分子。据此建立微球-微乳液、微孔-芯片的单分子信号放大方法，实现血浆ctDNA的高灵敏（单分子）、高特异（单碱基）、无污染（信号放大）和低成本（通用FRET探针）数字化检测；并通过调整两种荧光探针的混合比例，构建多重靶标的编码新方法，实现多个靶标的并行检测。本课题将为肿瘤精准治疗提供一个有力工具。

中文关键词： 循环肿瘤DNA；基因变异；单分子信号放大反应；数字化检测

英文摘要： The use of biopsy specimens is limited, but circulating tumor DNA allows for serial sampling during the course of treatment; thus it is important to detect ctDNA for individualized medicine of cancer. As the ultra low concentration of ctDNA, it is very difficult to quantify ctDNA in plasma. Digital PCR was successfully employed for the detection of ctDNA as its accurate quantification capability. However, the fluorescent probes used in digital PCR is non-universal and non-specific; and most importantly, it is easy to cause false-positive results due to the use of chip or flow cytometer for open-tube detection of PCR products. To overcome these problems, combination of signal amplification with digital counting is proposed. At first, a template of interest is amplified by a structure-specific enzymatic-reaction catalyzed by nuclease, yielding amplified oligonucleotide-signals. Then these signals are further amplified by a universal FRET modified-hairpin probe-mediated invader assay to produce amplified fluorescent signals. Based on this strategy, methods for signal amplification of single molecules are developed by using beads-emulsion PCR, and microchamber-chip PCR. Digital counting of ctDNA in plasma is thus achieved with a high sensitivity (single molecules),a high specificity (single base),a low contamination risk (signal amplification) and a low cost (universal FRET probes). More over, multiplex quantification is realized by coding multiple targets with different ratios between two dye-labeled FRET labels. The new method should supply a useful tool for precise cancer-treatment.

英文关键词： Circulating tumor DNA;Genetic variation;Single-molecule signal amplification;Digital Detection;Multiplex Quantification