發(fā)布時(shí)間：2018-05-27 12:00

  本文選題：高分辨率熔解曲線 + 線粒體DNA　； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:線粒體12SrRNA基因是母系遺傳性耳聾的突變熱點(diǎn)區(qū)域之一,本研究建立一種基于PCR-HRM技術(shù)的藥物性耳聾相關(guān)線粒體12SrRNA基因1494 CT和1555AG突變快速檢測方法。方法:我們主要針對(duì)目標(biāo)位點(diǎn)設(shè)計(jì)特異性的引物,通過飽和熒光染料進(jìn)行解鏈分析。首先采用定點(diǎn)誘變克隆策略構(gòu)建突變質(zhì)粒DNA標(biāo)準(zhǔn)品,再建立突變位點(diǎn)靶序列PCR-HRM分析體系,并對(duì)體系進(jìn)行一系列優(yōu)化,最后通過對(duì)106例非綜合征耳聾患者標(biāo)本進(jìn)行盲法分析評(píng)價(jià),同時(shí)以DNA直接測序法進(jìn)行驗(yàn)證。結(jié)果:建立的PCR-HRM檢測方法能準(zhǔn)確檢出線粒體12SrRNA基因1494 CT和1555AG突變,體系的穩(wěn)定性好、靈敏度高,各基因型的熔解曲線特征明顯且易于分析判斷;106例非綜合征耳聾患者標(biāo)本中檢出6例1555 AG突變,檢測結(jié)果與DNA測序結(jié)果一致。結(jié)論:建立了藥物性耳聾相關(guān)人類線粒體12SrRNA基因1494 CT和1555 AG突變的PCR-HRM檢測方法,該方法操作簡單快速、結(jié)果準(zhǔn)確可靠,可應(yīng)用于人群篩查和臨床常規(guī)分子診斷。某些遺傳病的致病基因與全基因組范圍內(nèi)的一些基因序列普遍存在高度同源性,這可造成基因點(diǎn)突變檢測結(jié)果的假陰性。最經(jīng)典的例子是由于α珠蛋白基因突變導(dǎo)致的α-地中海貧血,地中海貧血是全球最常見、對(duì)人類健康影響最大的單基因遺傳病之一,我國南方是α-地貧高發(fā)地區(qū)。α珠蛋白基因點(diǎn)突變主要發(fā)生在α2基因上,但α2珠蛋白基因與α1珠蛋白基因同源性高達(dá)97%,這容易造成α2珠蛋白基因點(diǎn)突變檢測結(jié)果的假陰性,也是非缺失型α-地貧基因的檢測方法更新緩慢的主要原因。目前已在臨床上應(yīng)用的α2珠蛋白基因點(diǎn)突變檢測技術(shù)存在操作繁瑣、檢測通量低、成本高等諸多問題,不適用于大規(guī)模人群篩查及常規(guī)分子診斷。本研究主要基于巢式非對(duì)稱PCR結(jié)合熒光探針與解鏈分析策略,建立一種快速準(zhǔn)確檢測α2珠蛋白基因WS、QS、CS、CD30、CD31五種點(diǎn)突變的新方法,且有效解決高同源性造成假陰性的問題。首先設(shè)計(jì)特異性擴(kuò)增α2-珠蛋白基因及目的序列單鏈引物,對(duì)α2-珠蛋白基因高度富集,排除同源性α1-珠蛋白基因的影響,再針對(duì)目標(biāo)位點(diǎn)設(shè)計(jì)特異性探針,結(jié)合熔解曲線分析檢測點(diǎn)突變。然后通過對(duì)體系的引物結(jié)構(gòu)與組成、擴(kuò)增組分、反應(yīng)程序等一系列條件調(diào)整與優(yōu)化,建立一種高效解決高同源性問題及準(zhǔn)確檢測α2珠蛋白基因五種點(diǎn)突變的新方法,并收集1250例已確診的非缺失型α-地貧和正�；蛐蚲DNA標(biāo)本對(duì)體系進(jìn)行穩(wěn)定性、特異性、靈敏性、實(shí)用性等全面評(píng)價(jià)及通過盲法分析對(duì)體系進(jìn)行臨床應(yīng)用評(píng)價(jià)。最后以實(shí)驗(yàn)室自建檢測體系的形式制備成常規(guī)使用的檢測試劑盒,經(jīng)充分的臨床應(yīng)用后進(jìn)行產(chǎn)業(yè)化推廣和應(yīng)用。本研究的結(jié)果顯示,所設(shè)計(jì)的引物及探針能夠有效檢出相對(duì)應(yīng)的基因型,通過對(duì)檢測體系的一系列條件優(yōu)化及評(píng)價(jià),建立的新方法基于熒光PCR平臺(tái)快速準(zhǔn)確檢測非缺失型α-地貧。采用新方法檢測收集的1250例樣本,陽性率為11.52%,準(zhǔn)確率高達(dá)100%,檢測靈敏極高,對(duì)檢測模板要求低。與傳統(tǒng)的檢測方法相比,該新方法具有操作簡單,省時(shí)、判斷結(jié)果直觀、實(shí)用性強(qiáng)、高效解決假陰性問題等優(yōu)勢(shì)。因此,本研究建立了基于巢式非對(duì)稱PCR結(jié)合熒光探針雜交與解鏈分析策略快速準(zhǔn)確檢測五種非缺失型α-地貧新方法,能夠有效解決高同源性問題,且通量高、成本低、滿足大規(guī)模人群篩查及常規(guī)分子診斷的方法學(xué)需要,且可為其他基因點(diǎn)突變疾病提供新的檢測方法思路。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R764.43

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本文編號(hào)：1941956