金磁微粒层析法结合直扩ARMS-PCR对MTHFR C677T基因多态性检测方法的建立及其临床应用

发布时间：2018-02-11 11:34

本文关键词： 金磁微粒层析全血直扩口腔拭子 MTHFR　出处：《西北大学》2016年硕士论文　论文类型：学位论文

【摘要】：分子生物基因组学研究需要高通量、低成本、可靠的生物样品采集方法和处理的方法。基因检测传统方法常依靠纯化的DNA样本,但是往往纯化DNA的过程耗时,成本较高,步骤复杂,且每一步过程中有产生交叉污染的可能,本研究基于PCR-金磁微粒层析法建立了一种直接采用全血或口腔拭子样本,无需提取基因组DNA,通过ARMS-PCR扩增进行基因分型的方法(简称直扩),对亚甲基四氢叶酸还原酶蛋白编码基因(MTHFR)C677T多态性进行检测。特异性的区分为3种基因型,即CC型(野生型),CT型(杂合突变型),和TT型(纯合突变型)。MTHFR蛋白是叶酸代谢等中至关重要的酶,它活性的变化会引起同型半胱氨酸升高等从而导致多种遗传性疾病的发生。因此建立一种检测MTHFR C677T基因多态性的方法是很有意义的。本方法检测样本种类为临床使用广泛的全血样本,及无创取样的口腔拭子样本,仅对样本做简单预处理即可直接进行PCR扩增分型,省去提取核酸的繁杂步骤,且基于金磁微粒层析系统,扩增采用防污染UNG酶体系,扩增完成后只需10分钟就可以得到目视化结果,总时间仅为1小时30分钟。本检测方法经济,便捷,准确度高,无需大型仪器及专业人员。临床样本验证,全血样本200例,口腔拭子样本50例,准确率均为100%。我们将在此基础上开发应用于更多的基因多态性研究,及疾病研究的基因分型产品,有望在临床上广泛应用。
[Abstract]:Molecular biogenomics requires high throughput, low cost and reliable methods for collecting and processing biological samples. Traditional methods for gene detection often rely on purified DNA samples, but the process of DNA purification is often time-consuming and costly. The steps are complicated, and there is the possibility of cross contamination in each step. In this study, a direct blood or oral swab sample was established based on PCR- gold magnetic particle chromatography. The polymorphism of methylenetetrahydrofolate reductase protein coding gene (MTHFR- C677T) was detected by ARMS-PCR amplification without extraction of genomic DNA.These genotypes were specifically classified into three genotypes. In other words, CC (wild type) CT (heterozygous mutant) and TT (homozygous mutants). MTHFR protein is an important enzyme in folic acid metabolism. The change of its activity will lead to the increase of homocysteine, which will lead to the occurrence of many kinds of hereditary diseases. Therefore, it is meaningful to establish a method to detect the polymorphism of MTHFR C677T gene. Using a wide range of whole blood samples, And non-invasive oral swab samples, PCR amplification and typing can be carried out directly by simply pretreating the samples, eliminating the complicated steps of extracting nucleic acids, and using the anti-contamination UNG enzyme system based on the gold magnetic particle chromatography system. It only takes 10 minutes to obtain visual results after amplification, and the total time is only 1 hour and 30 minutes. This method is economical, convenient, accurate, and does not need large instruments and professionals. Oral swabs were collected in 50 cases with the accuracy of 100. We will develop and apply to more gene polymorphism studies and genotyping products for disease research, which is expected to be widely used in clinical practice.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R440

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