琼脂糖微液滴数字PCR用于血液循环DNA多重突变检测的研究

Sensitive detection and accurate quantification of mutations in circulating DNA in blood are valuable for early screening, treatment management and prognosis of most cancers. However, due to the moderate sensitivity and accuracy of mutation detection methods currently used, sensitive and accurate detection of disease-related mutations in circulating DNA is very difficult, which hinders its further investigation and potential applications in the clinic. Microfluidics-based droplet digital PCR (ddPCR), which can quantify the absolute copy number of target nucleic acid, is particularly suitable for detection and quantification of rare mutations. Here we would develop a microfluidic-based argarose droplet digital PCR format and then apply it to amplify circulating DNA from patient blood sample. In the process of amplification, asymmetric PCR is implemented to obtain single stranded products, which are then covalently fixed in the argarose droplet through acrylamide polymerization. A set of fluorescently labeled probes are used to recognize and discriminate single nucleotide mutations in amplification products in argarose microsphere. In combination with the capability of multi-color fluorescence detection and very high detection speed of flow cytometer, it is feasible to detection multiple mutations (at most 28 mutations can be detected simultaneously) in a fast and accurate way, which may promote the applications of circulating DNA in the clinic.

血液循环DNA突变的检测和定量分析对肿瘤的早期筛查、疗效观察、预后评估等多方面均具有巨大的应用价值。限于现有方法的灵敏度或准确性，难以对血液循环DNA中与疾病相关的突变进行灵敏、准确检测，影响其研究和临床应用。微流控芯片液滴数字PCR（ddPCR）可绝对定量待测核酸，特别适用于微量核酸的检测和定量分析。本研究拟采用微流控芯片构建基于琼脂糖的液滴数字PCR，应用琼脂糖ddPCR扩增血液循环DNA，扩增过程引入不对称PCR生成单链DNA扩增产物；通过丙烯酰胺聚合反应将扩增产物共价固定于琼脂糖液滴内部，利用荧光探针识别琼脂糖液滴固化所得微球内部扩增产物并检测单碱基突变，结合流式细胞仪的多色荧光和高通量检测及准确计数能力，实现血液循环DNA突变的多重（可同时检测22种突变）、快速、准确检测，为探讨血液循环DNA突变与临床结果的关联提供支持。

现有的微滴数字PCR仪通常只能检测两种波长的荧光信号，不能满足临床上同时检测多靶标的需求，本课题从改进微滴数字PCR反应产物的检测方案和改变微滴状态及检测手段两方面入手，增加微滴数字PCR能够同时检测的靶标数量。首先通过使用与野生型序列相匹配的探针并结合染料的检测方法，可以同时检出与野生型序列不一致的多个突变序列；进而将低变性温度共扩增（COLD PCR）应用于微滴数字PCR，提高了基因突变检测的灵敏度；同时重点探索将双乳相微滴用于数字PCR的可行性，考查并优化了双乳相微滴生成芯片的设计和制作、热循环过程中双乳相微滴稳定性、防止双乳相微滴内水相的荧光信号向外水相的扩散，证明了双乳相微滴可以用于数字PCR。综合上述结果，本研究为探索采用流式细胞仪对数字PCR结果进行检测解决了技术上的可行性。项目资助待发表论文两篇。培养硕士生四名，其中两名已毕业，两名2018年毕业。项目投入经费23万元，支出20.9460万元，支出略有调整，各项支出与预算大致相符，剩余经费2.0540万元，剩余经费计划用于本项目研究后续支出。