双链引物探针介导的链置换反应在SNVs检测和多重PCR中的应用探索

The detection of single nucleotide variants (SNVs) and application of multiple-PCR play an important role in precision medicine initiative. The detection of SNVs and the construction of multiple-PCR encountered the following dilemma: the amplification of wild-type gene may consume amounts reaction substrates thus restrain the amplification of mutate-type; multiple point mutations usually span length and the use of multiple probes such as molecular beacon suffers design difficulties, poor compatibility or discrimination ability; the use of multiple primers were difficult in design and may formation dimmers and hairpin thus cannot ensure similar amplification efficiency for different targets. Aiming these problems and combine our previous research in strand displacement reaction, we creatively adopt double primer probe mediated allele specific PCR to restrain the amplification of wild-type gene thus realize the detection of rare SNVs; Utilize double strand probe mediated strand displacement reaction to solve the compatible dilemma thus enable the accurate detection of multiple SNVs in one target. Explore the application of double strand primer mediated multiple-PCR to realize the combination of primer and probe, reduce the complexity of reaction thus achieve the detection of multiple targets in one reaction. This project could provide technique support for clinical application for the detection of SNVs and multiple PCR. Moreover, it would promote the development of nucleic acid in vitro diagnostic research and product research.

单核苷酸变异(SNVs)的检测和多重PCR对精准医疗的实现具有重要的价值。目前，临床上常用SNVs检测和多重PCR存在以下问题：SNVs基因常伴随大量的野生基因干扰，野生基因的扩增会抑制突变基因的扩增；采用多个检测探针（如分子信标）检测多个SNVs位点，探针难于设计且体系相容性差；多重PCR体系多对引物很难保证多个靶基因相同的扩增效率。针对上述问题，我们创新性的采用双链引物探针介导的链置换反应来抑制野生型基因的扩增，从而实现稀有SNVs的检测；率先利用多个双链检测探针来解决多个探针难于相容的问题，实现多个SNVs的准确检测；探索双链引物介导的多重PCR实现引物和探针合一，减少体系的复杂程度实现多个靶基因的单反应体系同时检出。该项目的实施将为SNVs检测和多重PCR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。

单核苷酸变异(SNVs)的检测和多重PCR对精准医疗的实现具有重要的价值。目前，临床上常用SNVs检测和多重PCR存在以下问题：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；多重PCR体系多对引物很难保证多个靶基因相同的扩增效率。针对上述存在的问题，本课题在实施发过程中：(1)构建了一种基于toehold介导的链置换反应循环和分子信标介导的等温链置换聚合反应的超灵敏电化学miRNA生物传感器，实现10 aM~100 pM浓度范围内let 7a的准确灵敏检测；(2)构建了双链引物介导的链置换反应来检测SARS-COV-2的ORF、N、E基因。构建的多重荧光定量PCR体系对SARS-COV-2 ORF、N、E基因的检测限为400 copies/mL，检测时间约为2小时。多重双链引物探针方法设计简单、体系兼容性好，有望应用于多重PCR和SNPs检测等领域；(3)建立了一种简便高效的单链靶基因提取方法，实现具有杂交活性的长链靶基因在临床标本中的直接获取。并基于UDG介导的 PCR 技术合成具有杂交活性的长链检测探针。通过整合磁性纳米富集技术和 SDR，构建了能够准确快速检测 SNPs 的生物传感器。通过对 G6PD 缺乏症患者临床标本进行检测(c.1376G>T 和c.1388G>A 两个位点)，初步探索了检测平台在临床中的应用。

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