“分子開關(guān)”在HBV基因拉米夫定耐藥突變檢測中的應(yīng)用
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基因拉米夫定常見耐藥突變位點檢測中的應(yīng)用。 方法:文獻資料顯示乙肝患者最為常見的耐藥突變位點主要集中于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基因所有常見藥物耐藥突變位點的核酸序列,并克隆至PUC57載體得到包含HBV基因所有常見耐藥突變位點的突變模板1(204位耐藥突變?yōu)閅VDD);通過重疊延伸PCR方法定點突變突變模板1的204位使其突變?yōu)椋╕IDD)并將得到的PCR片段克隆至PMD-19T載體測序鑒定得到包含HBV所有常見耐藥突變位點的突變模板2(204位耐藥突變?yōu)閅IDD);提取HBV患者血漿游離DNA并設(shè)計特定引物,擴增得到野生型HBV基因片段,將其克隆至PMD19-T載體并經(jīng)測序鑒定得到野生模板。依據(jù)HBV基因序列設(shè)計針對M204V,M204I,L180M,V173L四個拉米夫定常見耐藥突變位點的突變檢測引物使其與突變模板配對而與野生模板不配對。對于突變檢測引物在其3’端及次3’端進行硫化修飾。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)分別對上述四個常見的耐藥突變位點進行PCR檢測。比較突變敏感分子開關(guān)對突變模板及野生模板的“開”及“關(guān)”作用,并通過對普通PCR及熒光定量PCR反應(yīng)條件及反應(yīng)體系的優(yōu)化,確定所建立的檢測平臺的檢測敏感度與檢測靈敏度。 結(jié)果:本課題成功建立了HBV基因拉米夫定四個常見藥物耐藥突變位點(M204V, M204I, V173L, L180M)的分子開關(guān)的突變檢測平臺。分子開關(guān)對于配對的突變模板可擴增出特異性產(chǎn)物,對于野生模板則無擴增產(chǎn)物或僅在很高拷貝數(shù)時出現(xiàn)。將突變模板及野生模板以10倍為單位逐級稀釋,獲得質(zhì)粒拷貝數(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)檢測平臺對于M204V的檢測靈敏度達到100拷貝,特異性達到1000拷貝;對M204I檢測靈敏度達100拷貝,檢測特異性高達105拷貝;對L180M檢測靈敏度達10拷貝,檢測特異性達1000拷貝,對V173L檢測靈敏度達10拷貝,檢測特異性達104拷貝。并且成功建立了HBV拉米夫定耐藥的多重PCR檢測平臺對上述四個檢測位點其檢測靈敏度達100拷貝,檢測特異性達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),對已知突變的檢測有較高的靈敏度和特異性,,可以檢測微量的突變,本課題所構(gòu)建的突變敏感分子開關(guān)檢測體系可以檢測拉米夫定治療的HBV患者微量的耐藥突變,對于乙肝病人臨床用藥指導(dǎo)及個體治療方案的調(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)位級別】:碩士
【學(xué)位授予年份】:2014
【分類號】:R440;R512.62
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