基因突變敏感性分子開關檢測HIV-1耐藥基因突變
發(fā)布時間：2018-10-21 10:16
【摘要】：目的：利用高保真聚合酶介導的突變敏感性分子開關，建立對HIV-1七個耐藥突變位進行快速篩查的技術平臺，采用單一PCR和多重PCR相結合，檢測耐藥基因相關突變的有無，為HIV耐藥性評估提供依據(jù)，指導HIV患者合理用藥。 方法：將包含HIV-1耐藥突變的PCR產(chǎn)物克隆至pMD-19-T載體，通過含氨芐的LB平板篩選陽性克隆，挑選菌落進行培養(yǎng)并提取質(zhì)粒DNA。對質(zhì)粒DNA進行PCR擴增初步確定陽性克隆，并通過測序分析確證，獲得包含HIV-1耐藥突變相對應的野生型質(zhì)粒模板。突變型模板直接由公司合成，包含目前已知的HIV-1耐藥突變二十七個，本研究選擇七個高頻突變位點建立快速檢測體系。所選七個突變位點分別是M41L(ATG/CTG)、K65R(AAA/AGA)、T69N(ACT/AAT)、M184V(ATG/GTG)、V75I(GTA/ATA)、V82A(GTC/GCC)和L90M(TTG/ATG)。實驗以這七個突變位點為檢測靶點，分別設計3’末端與野生型基因位點或突變基因位點配對的引物，硫化磷酸修飾3’末端并偶聯(lián)高保真DNA聚合酶構成的基因突變敏感性分子開關。首先在不同體系分別對含有相應七個突變的突變質(zhì)粒模板及野生型質(zhì)粒模板進行引物延伸反應，并通過凝膠成像系統(tǒng)對其進行分析；然后在同一體系同時對含有相應七個突變的突變質(zhì)粒模板及野生型質(zhì)粒模板進行引物延伸反應，并通過凝膠成像系統(tǒng)對其進行分析；最后結合熒光定量分析進行檢測。 結果：在不同反應體系分別對七個熱點突變進行檢測。在一定的反應條件及反應體系下，特異性野生檢測引物與野生模板配對得以延伸；而與突變模板不配對則不能延伸。當特異性突變檢測引物與突變模板配對，引物得以延伸；而與野生模板不配對則不能延伸。結果顯示，使用野生型質(zhì)粒模板，該方法僅能使野生型等位基因相關引物得以延伸，而突變等位基因位點特異性引物不能被延伸。反之，使用突變質(zhì)粒模板，該方法僅能使突變型等位基因位點相關引物得以延伸，而野生型等位基因位點特異性引物不能被延伸。在同一反應體系同時對以上熱點突變進行檢測。同樣，完全配對引物能被延伸，不完全配對引物不能被延伸。分子開關對上述七個位點的識別敏感性大部分可達到10~1~10~9拷貝，特異性分別為10~4~10~5拷貝，這一特定引物擴增特定模板的特異性在突變與野生序列之間的達到3個或3個以上對數(shù)級，能有效早期檢測耐藥突變。 結論：高保真酶介導的突變敏感性分子開關可用于已知基因突變的檢測，，除可用于檢測某些遺傳性突變外，還可以檢測耐藥基因突變。高保真DNA聚合酶偶聯(lián)硫化修飾引物構成的突變敏感性分子開關能夠快速篩查HIV-1七種突變，該技術在HIV-1耐藥突變檢測具有較大的潛在應用價值，可用來指導用藥，尤其是早期監(jiān)控耐藥基因的突變。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R440;R512.91
本文編號：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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R440;R512.91
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