基于四引物ARMS-PCR技术的G6PD缺陷症6个热点突变的检测方法的建立与评价

发布时间：2018-04-11 01:38

本文选题：G6PD缺陷症 + 纯合子　；参考：《重庆医科大学》2015年硕士论文

【摘要】：目的葡萄糖。6-磷酸脱氢酶(Glucose-6-phosphatedehy DrogenaseG6PD)缺陷症是一种最常见的遗传性红细胞酶缺陷病。G6PD基因突变是G6PD缺陷症最主要的原因。而目前已有的一些基因突变的检测方法要么操作复杂、耗时长、费用昂贵,要么不能一次性区分杂合与纯合,使在临床的推广使用受到限制。而四引物扩增受阻突变体系聚合酶链反应(Tetra-primer Amplification Refractory Mutation System-Polymerase Chain Reaction, Tetra-primer ARMS-PCR)具有简便、迅速、准确度好、特异性高并且能够一次性区分等位基因是否纯合的优点。本研究诣在建立一种四引物扩增受阻突变体系PCR快速检测中国人G6PD缺陷症6种高频突变位点G1388A、G1376T、A95G、 C10041、 C1024T、G871A的方法。方法1.针对中国人群G6PD基因G1388A、G1376T、A95G, C1004T、 C1024T、G871A6个常见的突变位点分别构建相应的DNA阳性参考品。按照T-ARMS-PCR引物设计原则分别设计G6PD基因6种高频突变位点的优化引物。2.通过对退火温度及内外引物浓度比的优化建立针对G6PD基因以上6个突变位点的T-ARMS-PCR方法。3.收集G6PD缺陷症标本(已用ARMS-PCR方法确诊有G1 388A、G1376T、A95G突变)和疑似G6PD缺陷症标本。用成功建立的T=ARMS-PCR方法分别检测89例G1388A位点突变的G6PD缺陷症患者和50例正常对照样本中G6PD基因G1388A位点,67例G1376T位点突变的G6PD缺陷症患者和50例正常对照样本中G6PD基因G1376T位点,38例A95G位点突变的G6PD缺陷症患者和50例正常对照样本中G6PD基因A95G位点。以及检测245例G6PD疑似患者标本G6PD基因C1004T、C1024T、 G871A三个位点。并用DNA测序进行验证。结果1.成功构建了G1388A、G1376T、A95G、C1004T、C1024T、G871A位点的阳性参考品以及设计了T-ARMS-PCR优化引物。2.成功建立了检测G6PD基因G1388A、G1376T、A95G、C1004T、 C1024T、G871A6个位点的T-ARMS-PCR方法。3. T-ARMS-PCR方法检测89例G1388A位点突变的G6PD缺陷症患者,检测出80例杂合子突变,9例杂合子突变,与测序结果一致；67例G1376T位点突变的G6PD缺陷症患者,检测出56例纯合子突变,11例杂合子突变,与测序结果一致。38例A95G位点突变的G6PD缺陷症患者,检测出35例纯合子突变,3例杂合子突变,与测序结果一致。245例G6PD疑似患者中检测出13例G871A位点突变,其中6例为杂合子突变。16例C1024T突变,其中5例为杂合子突变。结论成功的建立了一种检测G6PD缺陷症相关的突变位点的方法,并且不需要特殊的仪器设备。这种方法只通过6管PCR反应就可以检测G6PD缺陷症6种常见的突变。在不久的将来,在基于对G6PD缺陷症筛查的流行病学研究的基础上,可以应用于大量临床样本的检测。
[Abstract]:Glucose-6-phosphate dehydrogenase (Glucose-6-phosphatedehy G6PDD) deficiency is the most common genetic erythrocyte enzyme deficiency disease. G6PD gene mutation is the main cause of G6PD deficiency.However, some existing detection methods for gene mutations are either complicated, time-consuming and expensive, or can not distinguish heterozygosity from homozygosity at one time.However, Tetra-primer Amplification Refractory Mutation System-Polymerase Chain reaction (Tetra-primer ARMS-PCR) has the advantages of simplicity, rapidity, accuracy, specificity and the ability to distinguish homozygous alleles at once.The aim of this study was to establish a four-primer amplified blocked mutation system (PCR) for the rapid detection of G1388A, C10041 and C1024TnG871A in Chinese patients with G6PD deficiency at six high frequency mutation sites (G1388A, G1376TnA95G, C10041, C1024TG871A).Method 1.The corresponding DNA positive reference materials were constructed for 6 common mutation sites of G6PD gene G1388A, C1004T, C1024TG871A in Chinese population.According to the principle of T-ARMS-PCR primer design, the optimized primers. 2. 2 for 6 high frequency mutation sites of G6PD gene were designed respectively.By optimizing the annealing temperature and the ratio of internal and external primer concentration, the T-ARMS-PCR method for the above 6 mutation sites of G6PD gene was established.The specimens of G6PD deficiency (G 1 388 Agna G1376TN A95G mutation) and suspected G6PD deficiency were collected.The successfully established T=ARMS-PCR method was used to detect G6PD gene in 89 patients with G1388A locus mutation G6PD deficiency and 50 normal controls in 67 patients with G1388A locus mutation G6PD deficiency and 50 normal controls.The A95G locus of G6PD gene was found in 38 patients with A95G mutation in G1376T locus and 50 normal controls.Three loci of G6PD gene, C1004 TX C1024T and G871A, were detected in 245 suspected G6PD patients.The results were verified by DNA sequencing.Result 1.The positive reference material of G1388A, G1376T, A95GN, C1004, C1024TnG871A, and the optimized primer of T-ARMS-PCR, .2. were constructed successfully.A T-ARMS-PCR method for the detection of C1004T, C1024TG871A6 loci of G6PD gene G1388A was successfully established.T-ARMS-PCR method was used to detect G1388A mutation in 89 patients with G6PD deficiency, 80 heterozygote mutations were detected in 9 heterozygotes, and 67 patients with G1376T mutation were detected by T-ARMS-PCR.56 cases of homozygote mutation and 11 cases of heterozygote mutation were detected. The results of sequencing were consistent with those of 38 patients with G6PD deficiency with A95G mutation. 35 cases of homozygote mutation were detected in 3 cases of heterozygote mutation.In accordance with the sequencing results, 13 cases of G871A locus mutation were detected among the 245 suspected G6PD patients, among which 6 cases were heterozygous mutations. 16 cases were C1024T mutations, 5 cases were heterozygote mutations.Conclusion A method for the detection of mutation sites associated with G6PD deficiency has been successfully established, and no special equipment is required.This method can detect 6 common mutations in G6PD deficiency by only 6 tube PCR reaction.In the near future, based on epidemiological studies of G6PD deficiency screening, it can be applied to a large number of clinical samples.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R440

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