本文選題：HBV + 耐藥突變��； 參考：《蘇州大學(xué)》2014年碩士論文

【摘要】：目的：研究硫化修飾引物和高保真DNA聚合酶介導(dǎo)的分子開關(guān)在HBV基因拉米夫定常見(jiàn)耐藥突變位點(diǎn)檢測(cè)中的應(yīng)用。 方法：文獻(xiàn)資料顯示乙肝患者最為常見(jiàn)的耐藥突變位點(diǎn)主要集中于HBV基因的pol聚合酶基中一段500bp的核酸序列之內(nèi)。人工合成包含HBV基因所有常見(jiàn)藥物耐藥突變位點(diǎn)的核酸序列，并克隆至PUC57載體得到包含HBV基因所有常見(jiàn)耐藥突變位點(diǎn)的突變模板1（204位耐藥突變?yōu)閅VDD）；通過(guò)重疊延伸PCR方法定點(diǎn)突變突變模板1的204位使其突變?yōu)椋╕IDD）并將得到的PCR片段克隆至PMD-19T載體測(cè)序鑒定得到包含HBV所有常見(jiàn)耐藥突變位點(diǎn)的突變模板2（204位耐藥突變?yōu)閅IDD）；提取HBV患者血漿游離DNA并設(shè)計(jì)特定引物，擴(kuò)增得到野生型HBV基因片段，將其克隆至PMD19-T載體并經(jīng)測(cè)序鑒定得到野生模板。依據(jù)HBV基因序列設(shè)計(jì)針對(duì)M204V，M204I，L180M，V173L四個(gè)拉米夫定常見(jiàn)耐藥突變位點(diǎn)的突變檢測(cè)引物使其與突變模板配對(duì)而與野生模板不配對(duì)。對(duì)于突變檢測(cè)引物在其3’端及次3’端進(jìn)行硫化修飾。以高保真DNA聚合酶及硫化修飾的引物所構(gòu)成的突變敏感分子開關(guān)分別對(duì)上述四個(gè)常見(jiàn)的耐藥突變位點(diǎn)進(jìn)行PCR檢測(cè)。比較突變敏感分子開關(guān)對(duì)突變模板及野生模板的“開”及“關(guān)”作用，并通過(guò)對(duì)普通PCR及熒光定量PCR反應(yīng)條件及反應(yīng)體系的優(yōu)化，確定所建立的檢測(cè)平臺(tái)的檢測(cè)敏感度與檢測(cè)靈敏度。 結(jié)果：本課題成功建立了HBV基因拉米夫定四個(gè)常見(jiàn)藥物耐藥突變位點(diǎn)（M204V, M204I, V173L, L180M）的分子開關(guān)的突變檢測(cè)平臺(tái)。分子開關(guān)對(duì)于配對(duì)的突變模板可擴(kuò)增出特異性產(chǎn)物，對(duì)于野生模板則無(wú)擴(kuò)增產(chǎn)物或僅在很高拷貝數(shù)時(shí)出現(xiàn)。將突變模板及野生模板以10倍為單位逐級(jí)稀釋，獲得質(zhì)�？截悢�(shù)為107-101的不同濃度。本課題所建立的突變敏感分子開關(guān)檢測(cè)平臺(tái)對(duì)于M204V的檢測(cè)靈敏度達(dá)到100拷貝，特異性達(dá)到1000拷貝；對(duì)M204I檢測(cè)靈敏度達(dá)100拷貝，檢測(cè)特異性高達(dá)105拷貝；對(duì)L180M檢測(cè)靈敏度達(dá)10拷貝，檢測(cè)特異性達(dá)1000拷貝，對(duì)V173L檢測(cè)靈敏度達(dá)10拷貝，檢測(cè)特異性達(dá)104拷貝。并且成功建立了HBV拉米夫定耐藥的多重PCR檢測(cè)平臺(tái)對(duì)上述四個(gè)檢測(cè)位點(diǎn)其檢測(cè)靈敏度達(dá)100拷貝，檢測(cè)特異性達(dá)1000拷貝。 結(jié)論：高保真DNA聚合酶介導(dǎo)的突變敏感分子開關(guān)，對(duì)已知突變的檢測(cè)有較高的靈敏度和特異性，，可以檢測(cè)微量的突變，本課題所構(gòu)建的突變敏感分子開關(guān)檢測(cè)體系可以檢測(cè)拉米夫定治療的HBV患者微量的耐藥突變，對(duì)于乙肝病人臨床用藥指導(dǎo)及個(gè)體治療方案的調(diào)整有重要的意義。
[Abstract]:Aim: to study the application of vulcanized modified primers and high fidelity DNA polymerase mediated molecular switches in detection of common drug resistance mutation sites of HBV gene lamivudine.Methods: literature showed that the most common drug resistance mutation sites in hepatitis B patients were mainly in the nucleic acid sequence of a segment of 500bp in the pol polymerase of HBV gene.Synthesis of nucleic acid sequences containing all common drug resistance mutation sites in the HBV gene,And cloned into PUC57 vector to obtain mutation template containing all common drug resistance mutation sites of HBV gene. The mutation of drug resistance at position 1 is YVDDD. By overlapping extension PCR method, point mutation template 1 is mutated to YIDD and PCR is obtained.The fragment was cloned into PMD-19T vector and sequenced to identify the mutation template containing all common drug resistant mutation sites of HBV. The mutation was identified as YIDD. Free DNA was extracted from the plasma of HBV patients and specific primers were designed.The wild type HBV gene fragment was amplified and cloned into PMD19-T vector.According to the HBV gene sequence, the mutation detection primers were designed for four common drug resistant mutants of M204VX M204IN L180MnV173L so that they were paired with the mutation template but not matched with the wild template.The mutagenesis detection primer was vulcanized at the 3 'end and the second 3' end of the primer.The mutagenic sensitive molecular switches composed of high fidelity DNA polymerase and vulcanized primers were used to detect the four common drug resistance mutation sites by PCR.To compare the "on" and "off" effects of mutants sensitive molecular switches on mutation templates and wild templates, and to optimize the reaction conditions and reaction systems of common PCR and fluorescent quantitative PCR.Determine the detection sensitivity and sensitivity of the established test platform.Results: in this study, the molecular switch detection platform of four common drug resistance mutation sites of HBV gene, M204V, M204I, V173L, L180M, was successfully established.Molecular switches can amplify specific products for paired mutant templates, but no amplification products for wild templates or only appear at high copy numbers.Different concentrations of plasmid copy number 107-101 were obtained by diluting the mutant template and the wild template by 10 times unit step by step.The sensitivity of M204V is 100 copies, the specificity is 1000 copies, the sensitivity of M204I is 100 copies, the detection specificity of M204I is up to 105copies, the detection sensitivity of M204I is 100 copies, and the sensitivity of M204I is 1000 copies.The sensitivity for L180M was 10 copies, the detection specificity was 1000 copies, the sensitivity for V173L was 10 copies and the detection specificity was 104 copies.The multiplex PCR platform for HBV lamivudine resistance was successfully established. The sensitivity and specificity of the four sites were 100 copies and 1000 copies, respectively.Conclusion: the mutation sensitive molecular switch mediated by high fidelity DNA polymerase has high sensitivity and specificity for the detection of known mutations.The mutagenic sensitive molecular switch system constructed in this paper can detect minimal drug resistance mutations in patients with HBV treated with lamivudine, which is of great significance for clinical medication guidance and individual therapy of hepatitis B patients.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R440;R512.62

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 郭晏海,趙錦榮,崔大祥,閆小君;PCR-ELISA檢測(cè)血清中HBV聚合酶YMDD基因變異[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2002年04期

2 王虹,王省良,萬(wàn)成松,彭華國(guó),張文炳;微板核酸雜交-ELISA方法對(duì)肝炎病人血清中HBV DNA的定量檢測(cè)與分析[J];第一軍醫(yī)大學(xué)學(xué)報(bào);1999年04期

3 汪小娟;劉香萍;羅堯都;葉國(guó)強(qiáng);謝春英;;3種檢測(cè)乙型肝炎病毒YMDD變異方法比較及結(jié)果分析[J];中國(guó)感染控制雜志;2006年02期

4 曹衛(wèi),李文全;通用模板信號(hào)擴(kuò)增技術(shù)[J];國(guó)外醫(yī)學(xué).臨床生物化學(xué)與檢驗(yàn)學(xué)分冊(cè);2002年03期

5 彭翠英,張佳,郭紫芬,陳琳玲,廖端芳;SNP敏感性分子開關(guān)對(duì)神經(jīng)性耳聾GJB3中C→T突變點(diǎn)的識(shí)別[J];南華大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2003年02期

6 楊利黎;;乙型肝炎病毒YMDD變異的研究進(jìn)展[J];臨床和實(shí)驗(yàn)醫(yī)學(xué)雜志;2007年10期

7 汪榮生,張華,趙耘,朱玉芬,楊志軍;通用模板PCR技術(shù)快速檢測(cè)乙肝病毒YMDD變異株[J];實(shí)用臨床醫(yī)藥雜志;2005年01期

8 高秀麗,景奉香,楊劍波,趙建龍;單核苷酸多態(tài)性檢測(cè)分析技術(shù)[J];遺傳;2005年01期

9 張偉三 ,丁靜娟;HBV耐拉米夫定多聚酶基因變異檢測(cè)方法的建立[J];醫(yī)學(xué)文選;2002年03期

10 裴斐,鄭杰,寧鈞宇,由江峰,楊京平;乙型肝炎病毒DNA多聚酶基因YMDD突變的快速檢測(cè)[J];中華病理學(xué)雜志;2003年01期

本文編號(hào)：1757782