目标位点特异性探针PCR解链分析快速检测线粒体12S rRNA及α2珠蛋白基因点突变

发布时间：2018-05-27 12:00

本文选题：高分辨率熔解曲线 + 线粒体DNA　；参考：《南方医科大学》2017年硕士论文

【摘要】：目的:线粒体12SrRNA基因是母系遗传性耳聋的突变热点区域之一,本研究建立一种基于PCR-HRM技术的药物性耳聋相关线粒体12SrRNA基因1494 CT和1555AG突变快速检测方法。方法:我们主要针对目标位点设计特异性的引物,通过饱和荧光染料进行解链分析。首先采用定点诱变克隆策略构建突变质粒DNA标准品,再建立突变位点靶序列PCR-HRM分析体系,并对体系进行一系列优化,最后通过对106例非综合征耳聋患者标本进行盲法分析评价,同时以DNA直接测序法进行验证。结果:建立的PCR-HRM检测方法能准确检出线粒体12SrRNA基因1494 CT和1555AG突变,体系的稳定性好、灵敏度高,各基因型的熔解曲线特征明显且易于分析判断;106例非综合征耳聋患者标本中检出6例1555 AG突变,检测结果与DNA测序结果一致。结论:建立了药物性耳聋相关人类线粒体12SrRNA基因1494 CT和1555 AG突变的PCR-HRM检测方法,该方法操作简单快速、结果准确可靠,可应用于人群筛查和临床常规分子诊断。某些遗传病的致病基因与全基因组范围内的一些基因序列普遍存在高度同源性,这可造成基因点突变检测结果的假阴性。最经典的例子是由于α珠蛋白基因突变导致的α-地中海贫血,地中海贫血是全球最常见、对人类健康影响最大的单基因遗传病之一,我国南方是α-地贫高发地区。α珠蛋白基因点突变主要发生在α2基因上,但α2珠蛋白基因与α1珠蛋白基因同源性高达97%,这容易造成α2珠蛋白基因点突变检测结果的假阴性,也是非缺失型α-地贫基因的检测方法更新缓慢的主要原因。目前已在临床上应用的α2珠蛋白基因点突变检测技术存在操作繁琐、检测通量低、成本高等诸多问题,不适用于大规模人群筛查及常规分子诊断。本研究主要基于巢式非对称PCR结合荧光探针与解链分析策略,建立一种快速准确检测α2珠蛋白基因WS、QS、CS、CD30、CD31五种点突变的新方法,且有效解决高同源性造成假阴性的问题。首先设计特异性扩增α2-珠蛋白基因及目的序列单链引物,对α2-珠蛋白基因高度富集,排除同源性α1-珠蛋白基因的影响,再针对目标位点设计特异性探针,结合熔解曲线分析检测点突变。然后通过对体系的引物结构与组成、扩增组分、反应程序等一系列条件调整与优化,建立一种高效解决高同源性问题及准确检测α2珠蛋白基因五种点突变的新方法,并收集1250例已确诊的非缺失型α-地贫和正常基因型gDNA标本对体系进行稳定性、特异性、灵敏性、实用性等全面评价及通过盲法分析对体系进行临床应用评价。最后以实验室自建检测体系的形式制备成常规使用的检测试剂盒,经充分的临床应用后进行产业化推广和应用。本研究的结果显示,所设计的引物及探针能够有效检出相对应的基因型,通过对检测体系的一系列条件优化及评价,建立的新方法基于荧光PCR平台快速准确检测非缺失型α-地贫。采用新方法检测收集的1250例样本,阳性率为11.52%,准确率高达100%,检测灵敏极高,对检测模板要求低。与传统的检测方法相比,该新方法具有操作简单,省时、判断结果直观、实用性强、高效解决假阴性问题等优势。因此,本研究建立了基于巢式非对称PCR结合荧光探针杂交与解链分析策略快速准确检测五种非缺失型α-地贫新方法,能够有效解决高同源性问题,且通量高、成本低、满足大规模人群筛查及常规分子诊断的方法学需要,且可为其他基因点突变疾病提供新的检测方法思路。
[Abstract]:Objective: the mitochondrial 12SrRNA gene is one of the hot spots in the maternal hereditary deafness. In this study, a rapid detection method for the 1494 CT and 1555AG mutations of drug-related deafness related mitochondrial 12SrRNA gene based on PCR-HRM technique was established. Line solution chain analysis. First, the mutant plasmid DNA standard was constructed by the fixed-point mutation cloning strategy, and then the PCR-HRM analysis system of the target sequence of the mutant site was established, and a series of optimization were carried out. Finally, 106 cases of non syndrome deafness were evaluated by blind analysis and verified by DNA direct sequencing. The results were established. PCR-HRM detection method can accurately detect the mitochondrial 12SrRNA gene 1494 CT and 1555AG mutation, the stability of the system is good, the sensitivity is high, the characteristics of the fusion curves of various genotypes are obvious and easy to be analyzed. 106 cases of non syndrome deafness are detected in 6 cases of 1555 AG mutation, and the results are in accordance with the results of DNA sequencing. Conclusion: a drug was established. The method of PCR-HRM detection of human mitochondrial 12SrRNA gene 1494 CT and 1555 AG mutations in sexual deafness is simple and fast, accurate and reliable, and can be applied to population screening and clinical routine molecular diagnosis. The most classic example is the alpha thalassemia caused by the mutation of the alpha globin gene. Thalassemia is one of the most common monogenic diseases in the world, which has the greatest impact on human health. The south of China is a high incidence region of alpha poverty. The point mutation of alpha globin gene occurs mainly in the alpha 2 base. As a result, the homology of alpha 2 globin gene and alpha 1 globin gene is up to 97%, which can easily cause false negative of detection results of alpha 2 globin gene point mutation. It is also the main reason for the slow updating of the detection methods of the non deletion gene of alpha poor gene. The detection technique of alpha 2 globin gene point mutation has been operating cumbersome in clinical application. The detection of low flux and high cost is not suitable for large-scale population screening and conventional molecular diagnosis. This study is based on nested asymmetric PCR combined with fluorescence probe and chain analysis strategy to establish a new method for rapid and accurate detection of the five point mutations of alpha 2 globin gene, such as WS, QS, CS, CD30, CD31, and to effectively solve the high homology The problem of false negative. First, we designed specific amplification of alpha 2- globin gene and sequence single strand primers, enriched the alpha 2- globin gene, eliminated the effect of homologous alpha 1- globin gene, and then designed specific probes for target loci, combined with the fusion curve analysis to detect point mutation. A series of conditions are adjusted and optimized, such as composition, amplification components and reaction procedures, and a new method to efficiently solve the high homology problem and to detect the five point mutation of alpha 2 globin gene accurately, and to collect 1250 confirmed non missing alpha ground poor and normal genotype gDNA specimens for stability, specificity, sensitivity, and practical use. A comprehensive evaluation of sex and the evaluation of the clinical application of the system through blind analysis. Finally, a routine test kit was prepared in the form of a laboratory self built detection system, which is commercialization and application after full clinical application. The results of this study show that the designed primers and probes can be effectively detected. By optimizing and evaluating a series of conditions of the detection system, the new method is based on the fluorescence PCR platform to quickly and accurately detect the non missing alpha - ground poverty. The new method is used to detect 1250 samples, the positive rate is 11.52%, the accuracy is up to 100%, the detection sensitivity is very high, the requirement of the detection template is low. The new method has the advantages of simple operation, time saving, intuitionistic judgment, strong practicability and high efficiency in solving false negative problems. Therefore, this study has established a new method based on nested asymmetric PCR combined with fluorescence probe hybridization and chain analysis to quickly and accurately detect five new methods of non deletion alpha ground poverty, which can effectively solve the problem of high homology. It has high flux and low cost. It meets the needs of large-scale population screening and conventional molecular diagnosis, and provides a new way of thinking for other gene point mutation diseases.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R764.43

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