本文選題：G6PD缺陷癥 + 純合子　； 參考：《重慶醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目 的葡萄糖。6-磷酸脫氫酶(Glucose-6-phosphatedehy DrogenaseG6PD)缺陷癥是一種最常見(jiàn)的遺傳性紅細(xì)胞酶缺陷病。G6PD基因突變是G6PD缺陷癥最主要的原因。而目前已有的一些基因突變的檢測(cè)方法要么操作復(fù)雜、耗時(shí)長(zhǎng)、費(fèi)用昂貴,要么不能一次性區(qū)分雜合與純合,使在臨床的推廣使用受到限制。而四引物擴(kuò)增受阻突變體系聚合酶鏈反應(yīng)(Tetra-primer Amplification Refractory Mutation System-Polymerase Chain Reaction, Tetra-primer ARMS-PCR)具有簡(jiǎn)便、迅速、準(zhǔn)確度好、特異性高并且能夠一次性區(qū)分等位基因是否純合的優(yōu)點(diǎn)。本研究詣在建立一種四引物擴(kuò)增受阻突變體系PCR快速檢測(cè)中國(guó)人G6PD缺陷癥6種高頻突變位點(diǎn)G1388A、G1376T、A95G、 C10041、 C1024T、G871A的方法。方法1.針對(duì)中國(guó)人群G6PD基因G1388A、G1376T、A95G, C1004T、 C1024T、G871A6個(gè)常見(jiàn)的突變位點(diǎn)分別構(gòu)建相應(yīng)的DNA陽(yáng)性參考品。按照T-ARMS-PCR引物設(shè)計(jì)原則分別設(shè)計(jì)G6PD基因6種高頻突變位點(diǎn)的優(yōu)化引物。2.通過(guò)對(duì)退火溫度及內(nèi)外引物濃度比的優(yōu)化建立針對(duì)G6PD基因以上6個(gè)突變位點(diǎn)的T-ARMS-PCR方法。3.收集G6PD缺陷癥標(biāo)本(已用ARMS-PCR方法確診有G1 388A、G1376T、A95G突變)和疑似G6PD缺陷癥標(biāo)本。用成功建立的T=ARMS-PCR方法分別檢測(cè)89例G1388A位點(diǎn)突變的G6PD缺陷癥患者和50例正常對(duì)照樣本中G6PD基因G1388A位點(diǎn),67例G1376T位點(diǎn)突變的G6PD缺陷癥患者和50例正常對(duì)照樣本中G6PD基因G1376T位點(diǎn),38例A95G位點(diǎn)突變的G6PD缺陷癥患者和50例正常對(duì)照樣本中G6PD基因A95G位點(diǎn)。以及檢測(cè)245例G6PD疑似患者標(biāo)本G6PD基因C1004T、C1024T、 G871A三個(gè)位點(diǎn)。并用DNA測(cè)序進(jìn)行驗(yàn)證。結(jié)果1.成功構(gòu)建了G1388A、G1376T、A95G、C1004T、C1024T、G871A位點(diǎn)的陽(yáng)性參考品以及設(shè)計(jì)了T-ARMS-PCR優(yōu)化引物。2.成功建立了檢測(cè)G6PD基因G1388A、G1376T、A95G、C1004T、 C1024T、G871A6個(gè)位點(diǎn)的T-ARMS-PCR方法。3. T-ARMS-PCR方法檢測(cè)89例G1388A位點(diǎn)突變的G6PD缺陷癥患者,檢測(cè)出80例雜合子突變,9例雜合子突變,與測(cè)序結(jié)果一致；67例G1376T位點(diǎn)突變的G6PD缺陷癥患者,檢測(cè)出56例純合子突變,11例雜合子突變,與測(cè)序結(jié)果一致。38例A95G位點(diǎn)突變的G6PD缺陷癥患者,檢測(cè)出35例純合子突變,3例雜合子突變,與測(cè)序結(jié)果一致。245例G6PD疑似患者中檢測(cè)出13例G871A位點(diǎn)突變,其中6例為雜合子突變。16例C1024T突變,其中5例為雜合子突變。結(jié)論成功的建立了一種檢測(cè)G6PD缺陷癥相關(guān)的突變位點(diǎn)的方法,并且不需要特殊的儀器設(shè)備。這種方法只通過(guò)6管PCR反應(yīng)就可以檢測(cè)G6PD缺陷癥6種常見(jiàn)的突變。在不久的將來(lái),在基于對(duì)G6PD缺陷癥篩查的流行病學(xué)研究的基礎(chǔ)上,可以應(yīng)用于大量臨床樣本的檢測(cè)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R440

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