基因突变敏感性分子开关检测HIV-1耐药基因突变
发布时间：2018-10-21 10:16
【摘要】：目的：利用高保真聚合酶介导的突变敏感性分子开关，建立对HIV-1七个耐药突变位进行快速筛查的技术平台，采用单一PCR和多重PCR相结合，检测耐药基因相关突变的有无，为HIV耐药性评估提供依据，指导HIV患者合理用药。 方法：将包含HIV-1耐药突变的PCR产物克隆至pMD-19-T载体，通过含氨苄的LB平板筛选阳性克隆，挑选菌落进行培养并提取质粒DNA。对质粒DNA进行PCR扩增初步确定阳性克隆，并通过测序分析确证，获得包含HIV-1耐药突变相对应的野生型质粒模板。突变型模板直接由公司合成，包含目前已知的HIV-1耐药突变二十七个，本研究选择七个高频突变位点建立快速检测体系。所选七个突变位点分别是M41L(ATG/CTG)、K65R(AAA/AGA)、T69N(ACT/AAT)、M184V(ATG/GTG)、V75I(GTA/ATA)、V82A(GTC/GCC)和L90M(TTG/ATG)。实验以这七个突变位点为检测靶点，分别设计3’末端与野生型基因位点或突变基因位点配对的引物，硫化磷酸修饰3’末端并偶联高保真DNA聚合酶构成的基因突变敏感性分子开关。首先在不同体系分别对含有相应七个突变的突变质粒模板及野生型质粒模板进行引物延伸反应，并通过凝胶成像系统对其进行分析；然后在同一体系同时对含有相应七个突变的突变质粒模板及野生型质粒模板进行引物延伸反应，并通过凝胶成像系统对其进行分析；最后结合荧光定量分析进行检测。 结果：在不同反应体系分别对七个热点突变进行检测。在一定的反应条件及反应体系下，特异性野生检测引物与野生模板配对得以延伸；而与突变模板不配对则不能延伸。当特异性突变检测引物与突变模板配对，引物得以延伸；而与野生模板不配对则不能延伸。结果显示，使用野生型质粒模板，该方法仅能使野生型等位基因相关引物得以延伸，而突变等位基因位点特异性引物不能被延伸。反之，使用突变质粒模板，该方法仅能使突变型等位基因位点相关引物得以延伸，而野生型等位基因位点特异性引物不能被延伸。在同一反应体系同时对以上热点突变进行检测。同样，完全配对引物能被延伸，不完全配对引物不能被延伸。分子开关对上述七个位点的识别敏感性大部分可达到10~1~10~9拷贝，特异性分别为10~4~10~5拷贝，这一特定引物扩增特定模板的特异性在突变与野生序列之间的达到3个或3个以上对数级，能有效早期检测耐药突变。 结论：高保真酶介导的突变敏感性分子开关可用于已知基因突变的检测，，除可用于检测某些遗传性突变外，还可以检测耐药基因突变。高保真DNA聚合酶偶联硫化修饰引物构成的突变敏感性分子开关能够快速筛查HIV-1七种突变，该技术在HIV-1耐药突变检测具有较大的潜在应用价值，可用来指导用药，尤其是早期监控耐药基因的突变。
[Abstract]:Objective: to establish a rapid screening platform for seven drug resistance mutants of HIV-1 by using a high fidelity polymerase mediated mutation sensitivity molecular switch, and to detect the presence or absence of drug-resistant gene related mutations by combining a single PCR with multiple PCR. To provide the basis for the evaluation of drug resistance in HIV and to guide the rational use of drugs in patients with HIV. Methods: the PCR product containing drug-resistant mutation of HIV-1 was cloned into pMD-19-T vector. The positive clones were screened by LB plate containing ampicillin, the colony was selected for culture and the plasmid DNA. was extracted. The positive plasmid DNA was amplified by PCR and confirmed by sequencing. The wild type plasmid template containing HIV-1 resistance mutation was obtained. The mutant template was synthesized directly from the company and included 27 known mutations of HIV-1 resistance. In this study, seven high frequency mutation sites were selected to establish a rapid detection system. The seven mutation sites were M41L (ATG/CTG), K65R (AAA/AGA), T69N (ACT/AAT), M188V (ATG/GTG), V75I (GTA/ATA), V82A (GTC/GCC) and L90M (TTG/ATG). Using these seven mutation sites as the detection targets, we designed primers for 3 'terminal pairs with wild-type gene loci or mutant gene loci, respectively. Phosphoric acid sulphide modified 3 'terminal and coupled high fidelity DNA polymerase to construct gene mutation sensitive molecular switch. Firstly, the mutant plasmid template and wild-type plasmid template containing seven corresponding mutations were amplified by primer extension in different systems, and analyzed by gel imaging system. Then in the same system, the mutated plasmid template and the wild type plasmid template containing seven corresponding mutations were tested by primer extension and analyzed by gel imaging system. Finally, fluorescence quantitative analysis was used to detect the mutation plasmid template and the wild-type plasmid template. Results: seven hot spot mutations were detected in different reaction systems. Under certain reaction conditions and reaction system, the pair of specific wild detection primers and wild templates could be extended, but not matched with mutant templates. When the specific mutation detection primer is paired with the mutation template, the primer can be extended, but not with the wild template. The results showed that using wild type plasmid template, this method could only extend wild type allele related primers, but mutant allelic locus specific primers could not be extended. On the other hand, using mutant plasmid template, this method can only extend mutant allelic locus related primers, but wild-type allele locus specific primers can not be extended. The hot spot mutation was detected simultaneously in the same reaction system. Similarly, fully paired primers can be extended, and incomplete pairs of primers cannot be extended. The sensitivity of the molecular switch to the above seven loci was mostly 10 ~ 1 / 10 ~ (9) copies, with a specificity of 10 ~ 4 / 10 ~ 5 copies, respectively. The specificity of the specific template amplified by this specific primer was 3 or more logarithmic order between mutation and wild sequence. It can be used to detect drug resistance mutation in early stage. Conclusion: high fidelity enzyme mediated mutagenic molecular switches can be used for the detection of known gene mutations, as well as for the detection of some genetic mutations as well as drug resistance gene mutations. The mutagenic sensitive molecular switch composed of high fidelity DNA polymerase coupled vulcanized modified primers can quickly screen seven mutations of HIV-1. This technique has a great potential application value in the detection of drug resistance mutation of HIV-1 and can be used to guide drug use. In particular, early detection of mutations in drug resistance genes.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R440;R512.91
本文编号：2284800
[Abstract]:Objective: to establish a rapid screening platform for seven drug resistance mutants of HIV-1 by using a high fidelity polymerase mediated mutation sensitivity molecular switch, and to detect the presence or absence of drug-resistant gene related mutations by combining a single PCR with multiple PCR. To provide the basis for the evaluation of drug resistance in HIV and to guide the rational use of drugs in patients with HIV. Methods: the PCR product containing drug-resistant mutation of HIV-1 was cloned into pMD-19-T vector. The positive clones were screened by LB plate containing ampicillin, the colony was selected for culture and the plasmid DNA. was extracted. The positive plasmid DNA was amplified by PCR and confirmed by sequencing. The wild type plasmid template containing HIV-1 resistance mutation was obtained. The mutant template was synthesized directly from the company and included 27 known mutations of HIV-1 resistance. In this study, seven high frequency mutation sites were selected to establish a rapid detection system. The seven mutation sites were M41L (ATG/CTG), K65R (AAA/AGA), T69N (ACT/AAT), M188V (ATG/GTG), V75I (GTA/ATA), V82A (GTC/GCC) and L90M (TTG/ATG). Using these seven mutation sites as the detection targets, we designed primers for 3 'terminal pairs with wild-type gene loci or mutant gene loci, respectively. Phosphoric acid sulphide modified 3 'terminal and coupled high fidelity DNA polymerase to construct gene mutation sensitive molecular switch. Firstly, the mutant plasmid template and wild-type plasmid template containing seven corresponding mutations were amplified by primer extension in different systems, and analyzed by gel imaging system. Then in the same system, the mutated plasmid template and the wild type plasmid template containing seven corresponding mutations were tested by primer extension and analyzed by gel imaging system. Finally, fluorescence quantitative analysis was used to detect the mutation plasmid template and the wild-type plasmid template. Results: seven hot spot mutations were detected in different reaction systems. Under certain reaction conditions and reaction system, the pair of specific wild detection primers and wild templates could be extended, but not matched with mutant templates. When the specific mutation detection primer is paired with the mutation template, the primer can be extended, but not with the wild template. The results showed that using wild type plasmid template, this method could only extend wild type allele related primers, but mutant allelic locus specific primers could not be extended. On the other hand, using mutant plasmid template, this method can only extend mutant allelic locus related primers, but wild-type allele locus specific primers can not be extended. The hot spot mutation was detected simultaneously in the same reaction system. Similarly, fully paired primers can be extended, and incomplete pairs of primers cannot be extended. The sensitivity of the molecular switch to the above seven loci was mostly 10 ~ 1 / 10 ~ (9) copies, with a specificity of 10 ~ 4 / 10 ~ 5 copies, respectively. The specificity of the specific template amplified by this specific primer was 3 or more logarithmic order between mutation and wild sequence. It can be used to detect drug resistance mutation in early stage. Conclusion: high fidelity enzyme mediated mutagenic molecular switches can be used for the detection of known gene mutations, as well as for the detection of some genetic mutations as well as drug resistance gene mutations. The mutagenic sensitive molecular switch composed of high fidelity DNA polymerase coupled vulcanized modified primers can quickly screen seven mutations of HIV-1. This technique has a great potential application value in the detection of drug resistance mutation of HIV-1 and can be used to guide drug use. In particular, early detection of mutations in drug resistance genes.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R440;R512.91
【参考文献】
相关期刊论文 前5条
1 张超;范忠鹏;朱旺升;赵莹;陈绅波;李凯;周宇荀;肖君华;;应用改进的通用荧光PCR引物进行多重STR分型[J];动物学杂志;2008年03期
2 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
3 陈琳玲,张佳,彭翠英,廖端芳,李弘剑,高汉林,李凯;DNA聚合酶高保真机理的新发现及其在SNP分析中的应用[J];遗传;2005年02期
4 郭紫芬,陈琳玲,张佳,彭翠英,杨向东,张旭,何淑雅,廖端芳,李凯;聚合酶3′外切活性对3′硫化修饰引物聚合反应的影响[J];中华医学遗传学杂志;2003年04期
5 Niel T CONSTANTINE;William KABAT;Richard Y ZHAO;;Update on the laboratory diagnosis and monitoring of HIV infection[J];Cell Research;2005年Z1期
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