当前位置:主页 > 医学论文 > 消化疾病论文 >

“分子开关”在HBV基因拉米夫定耐药突变检测中的应用

发布时间:2018-04-16 07:03

  本文选题:HBV + 耐药突变 ; 参考:《苏州大学》2014年硕士论文


【摘要】:目的:研究硫化修饰引物和高保真DNA聚合酶介导的分子开关在HBV基因拉米夫定常见耐药突变位点检测中的应用。 方法:文献资料显示乙肝患者最为常见的耐药突变位点主要集中于HBV基因的pol聚合酶基中一段500bp的核酸序列之内。人工合成包含HBV基因所有常见药物耐药突变位点的核酸序列,并克隆至PUC57载体得到包含HBV基因所有常见耐药突变位点的突变模板1(204位耐药突变为YVDD);通过重叠延伸PCR方法定点突变突变模板1的204位使其突变为(YIDD)并将得到的PCR片段克隆至PMD-19T载体测序鉴定得到包含HBV所有常见耐药突变位点的突变模板2(204位耐药突变为YIDD);提取HBV患者血浆游离DNA并设计特定引物,扩增得到野生型HBV基因片段,将其克隆至PMD19-T载体并经测序鉴定得到野生模板。依据HBV基因序列设计针对M204V,M204I,L180M,V173L四个拉米夫定常见耐药突变位点的突变检测引物使其与突变模板配对而与野生模板不配对。对于突变检测引物在其3’端及次3’端进行硫化修饰。以高保真DNA聚合酶及硫化修饰的引物所构成的突变敏感分子开关分别对上述四个常见的耐药突变位点进行PCR检测。比较突变敏感分子开关对突变模板及野生模板的“开”及“关”作用,并通过对普通PCR及荧光定量PCR反应条件及反应体系的优化,确定所建立的检测平台的检测敏感度与检测灵敏度。 结果:本课题成功建立了HBV基因拉米夫定四个常见药物耐药突变位点(M204V, M204I, V173L, L180M)的分子开关的突变检测平台。分子开关对于配对的突变模板可扩增出特异性产物,对于野生模板则无扩增产物或仅在很高拷贝数时出现。将突变模板及野生模板以10倍为单位逐级稀释,获得质粒拷贝数为107-101的不同浓度。本课题所建立的突变敏感分子开关检测平台对于M204V的检测灵敏度达到100拷贝,特异性达到1000拷贝;对M204I检测灵敏度达100拷贝,检测特异性高达105拷贝;对L180M检测灵敏度达10拷贝,检测特异性达1000拷贝,对V173L检测灵敏度达10拷贝,检测特异性达104拷贝。并且成功建立了HBV拉米夫定耐药的多重PCR检测平台对上述四个检测位点其检测灵敏度达100拷贝,检测特异性达1000拷贝。 结论:高保真DNA聚合酶介导的突变敏感分子开关,对已知突变的检测有较高的灵敏度和特异性,,可以检测微量的突变,本课题所构建的突变敏感分子开关检测体系可以检测拉米夫定治疗的HBV患者微量的耐药突变,对于乙肝病人临床用药指导及个体治疗方案的调整有重要的意义。
[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.
【学位授予单位】:苏州大学
【学位级别】:硕士
【学位授予年份】:2014
【分类号】:R440;R512.62

【参考文献】

相关期刊论文 前10条

1 郭晏海,赵锦荣,崔大祥,闫小君;PCR-ELISA检测血清中HBV聚合酶YMDD基因变异[J];第四军医大学学报;2002年04期

2 王虹,王省良,万成松,彭华国,张文炳;微板核酸杂交-ELISA方法对肝炎病人血清中HBV DNA的定量检测与分析[J];第一军医大学学报;1999年04期

3 汪小娟;刘香萍;罗尧都;叶国强;谢春英;;3种检测乙型肝炎病毒YMDD变异方法比较及结果分析[J];中国感染控制杂志;2006年02期

4 曹卫,李文全;通用模板信号扩增技术[J];国外医学.临床生物化学与检验学分册;2002年03期

5 彭翠英,张佳,郭紫芬,陈琳玲,廖端芳;SNP敏感性分子开关对神经性耳聋GJB3中C→T突变点的识别[J];南华大学学报(医学版);2003年02期

6 杨利黎;;乙型肝炎病毒YMDD变异的研究进展[J];临床和实验医学杂志;2007年10期

7 汪荣生,张华,赵耘,朱玉芬,杨志军;通用模板PCR技术快速检测乙肝病毒YMDD变异株[J];实用临床医药杂志;2005年01期

8 高秀丽,景奉香,杨剑波,赵建龙;单核苷酸多态性检测分析技术[J];遗传;2005年01期

9 张伟三 ,丁静娟;HBV耐拉米夫定多聚酶基因变异检测方法的建立[J];医学文选;2002年03期

10 裴斐,郑杰,宁钧宇,由江峰,杨京平;乙型肝炎病毒DNA多聚酶基因YMDD突变的快速检测[J];中华病理学杂志;2003年01期



本文编号:1757782

资料下载
论文发表

本文链接:https://www.wllwen.com/yixuelunwen/xiaohjib/1757782.html


Copyright(c)文论论文网All Rights Reserved | 网站地图 |

版权申明:资料由用户06e2d***提供,本站仅收录摘要或目录,作者需要删除请E-mail邮箱bigeng88@qq.com