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

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.

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。

检测循环肿瘤DNA（ctDNA）已成为实现肿瘤无创实时检测的有效途径。然而，ctDNA在总循环游离DNA（cfDNA）中含量很低，并且其序列与正常DNA片段差异微小，因此要求检测方法具有极高的灵敏度与特异性。尽管数字化PCR技术可实现低丰度ctDNA片段（~0.01%）的检测，但因其基于模板扩增，极易造成交叉污染。为了避免这一问题，本课题提出了用信号扩增取代模板扩增构建核酸数字化检测的新策略。. 课题首先发展了基于微孔芯片与水凝胶微球芯片的数字化PCR定量检测方法，实现了对母血中胎儿染色体异常与粪便中结直肠癌相关甲基化基因的定量检测，为建立数字化信号扩增检测方法提供了依据。. 为构建数字化信号扩增方法，选取核酸侵入反应作为信号转化与放大的策略，并对其进行优化改造，提升信号扩增的特异性与灵敏度，来满足数字化检测的要求。在此过程中，发展了高特异核酸侵入反应探针设计方法，并以此建立了基于核酸侵入信号扩增的基因突变定量检测新方法，实现了对cfDNA中基因突变的高灵敏检测（0.1%），灵敏度比传统基于水解探针的荧光定量PCR法提高了10倍；将核酸侵入反应引入到数字化PCR中，发展了高灵敏数字化PCR检测新方法，可检测低至0.00001%的基因突变，比传统数字化PCR的灵敏度高1000倍；通过引入纳米金探针杂交反应，建立了基于核酸侵入反应的基因突变可视化检测方法，实现了对环介导等温扩增产物的单碱基分辨可视化检测，并创建了集高灵敏PCR扩增、高特异序列识别与可视化信号输出的基因突变单管闭管检测新方法（“管子实验室”），可对低至6拷贝且丰度仅为0.1%的突变模板进行肉眼可视化检测；为了降低核酸侵入信号扩增的背景信号，提出了基于可控延伸的低背景级联核酸侵入反应新原理，将传统级联核酸侵入信号扩增的灵敏度提高了40倍，并构建了个体化用药基因突变检测逻辑门。. 最终，将低背景级联核酸侵入信号扩增反应与微球微乳液扩增结合，创建了可实现单分子扩增检测的数字化信号扩增核酸检测新方法，为发展无扩增产物污染的核酸数字化检测奠定了基础。