基于UDG介导PCR、磁性纳米富集和链置换反应的SNPs准确快速检测方法

The detection of single-nucleotide polymorphisms (SNPs) plays an important role in precision medicine initiative. The rapid detection of SNPs encountered the following dilemma: it is difficult to obtain high copy single-stranded target gene with hybridization activity from the clinical samples; detection probe, the length of which matches with target gene, cannot be accurate synthesized as the restriction of nucleic acid chemical synthesis technology; DNA hybridization between target gene and detection probe requires optimization because this process does not strictly follow the Watson-Crick pairing rules. Aiming these problems, we put forward a new strategy to obtain single-stranded nucleic acid as target based on magnetic nanoparticles enrichment and PCR. This project creatively combined UDG with PCR to acquire long-length detection probe; explore the kinetics parameters of long-length strand displacement reaction to enhance analytical performance. The as prepared methods for single-strand target gene and long-length detection probe extraction is an innovation in nucleic acids point-of-care test. The developed biosensor based on UDG mediated PCR, magnetic nanoparticles enrichment, and strand displacement reaction for nucleic acid diagnosis is a original method. This project could provide technique support for clinical application of biosensor. Moreover, it would promote the development of nucleic acid in vitro diagnostic research and product research.

单核苷酸多态性(SNPs)检测在精准医疗中具有极其重要的价值。目前临床样本SNPs快速检测存在三个关键问题：难以提取具有杂交活性的高拷贝单链靶基因；受核酸化学合成技术限制，无法准确高效合成与靶基因长度相匹配的检测探针；核酸杂交反应不严格遵循碱基互补配对规则，不能保证核酸杂交特异性。针对上述问题，我们创新性耦合磁性纳米富集和PCR技术，建立临床样本中单链靶基因的提取方法；率先利用尿嘧啶DNA糖基化酶（UDG）介导PCR技术合成长链检测探针；探索长链核酸链置换反应的动力学特征，提高其检测性能。拟建立的单链靶基因提取方法和长链检测探针合成技术是临床样本核酸准确检测在理论和技术上的创新。基于UDG介导PCR、磁性纳米富集和链置换反应构建的生物传感技术平台用于临床样本中SNPs检测是原创性思路。该项目的实施将为生物传感器的临床应用提供全新技术平台，为开创核酸体外诊断研究新道路及系列产品研发奠定基础。

单核苷酸多态性（SNPs）在临床中具有重要的意义，其与疫病易感性、药物抗性等密切相关。本项目耦合磁性富集技术和传统 PCR 技术构建了一种适于临床的单链靶基因提取方法；利用UDG 介导的 PCR 技术，用于获得具有杂交活性且荧光标记的检测探针；结合上述方法我们建立了更简便、经济的 SNPs 准确快速检测平台，该平台与荧光分光光度计、荧光显微镜、荧光定量 PCR 仪等仪器兼容。基于已建立的平台，我们成功检测了与G6PD缺乏症相关的1376G>T突变。在此基础上，为更加特异地检测点突变，我们比较了不同链置换策略的性能，设计了一种新型Bulge-loop探针以及开发了一种基于模拟指导的纳米机器设计策略。本项目研究成果有望为临床中SNPs检测提供更加简便、通用的手段，为SNPs的特异检测提供设计指导，加速SNPs检测在临床中的应用。