基因突變敏感性分子開關(guān)檢測(cè)HIV-1耐藥基因突變
發(fā)布時(shí)間：2018-10-21 10:16
【摘要】：目的：利用高保真聚合酶介導(dǎo)的突變敏感性分子開關(guān)，建立對(duì)HIV-1七個(gè)耐藥突變位進(jìn)行快速篩查的技術(shù)平臺(tái)，采用單一PCR和多重PCR相結(jié)合，檢測(cè)耐藥基因相關(guān)突變的有無(wú)，為HIV耐藥性評(píng)估提供依據(jù)，指導(dǎo)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(guò)含氨芐的LB平板篩選陽(yáng)性克隆，挑選菌落進(jìn)行培養(yǎng)并提取質(zhì)粒DNA。對(duì)質(zhì)粒DNA進(jìn)行PCR擴(kuò)增初步確定陽(yáng)性克隆，并通過(guò)測(cè)序分析確證，獲得包含HIV-1耐藥突變相對(duì)應(yīng)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(gè)，本研究選擇七個(gè)高頻突變位點(diǎn)建立快速檢測(cè)體系。所選七個(gè)突變位點(diǎn)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(shí)驗(yàn)以這七個(gè)突變位點(diǎn)為檢測(cè)靶點(diǎn)，分別設(shè)計(jì)3’末端與野生型基因位點(diǎn)或突變基因位點(diǎn)配對(duì)的引物，硫化磷酸修飾3’末端并偶聯(lián)高保真DNA聚合酶構(gòu)成的基因突變敏感性分子開關(guān)。首先在不同體系分別對(duì)含有相應(yīng)七個(gè)突變的突變質(zhì)粒模板及野生型質(zhì)粒模板進(jìn)行引物延伸反應(yīng)，并通過(guò)凝膠成像系統(tǒng)對(duì)其進(jìn)行分析；然后在同一體系同時(shí)對(duì)含有相應(yīng)七個(gè)突變的突變質(zhì)粒模板及野生型質(zhì)粒模板進(jìn)行引物延伸反應(yīng)，并通過(guò)凝膠成像系統(tǒng)對(duì)其進(jìn)行分析；最后結(jié)合熒光定量分析進(jìn)行檢測(cè)。 結(jié)果：在不同反應(yīng)體系分別對(duì)七個(gè)熱點(diǎn)突變進(jìn)行檢測(cè)。在一定的反應(yīng)條件及反應(yīng)體系下，特異性野生檢測(cè)引物與野生模板配對(duì)得以延伸；而與突變模板不配對(duì)則不能延伸。當(dāng)特異性突變檢測(cè)引物與突變模板配對(duì)，引物得以延伸；而與野生模板不配對(duì)則不能延伸。結(jié)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(guān)引物得以延伸，而突變等位基因位點(diǎn)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(diǎn)相關(guān)引物得以延伸，而野生型等位基因位點(diǎn)特異性引物不能被延伸。在同一反應(yīng)體系同時(shí)對(duì)以上熱點(diǎn)突變進(jìn)行檢測(cè)。同樣，完全配對(duì)引物能被延伸，不完全配對(duì)引物不能被延伸。分子開關(guān)對(duì)上述七個(gè)位點(diǎn)的識(shí)別敏感性大部分可達(dá)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(kuò)增特定模板的特異性在突變與野生序列之間的達(dá)到3個(gè)或3個(gè)以上對(duì)數(shù)級(jí)，能有效早期檢測(cè)耐藥突變。 結(jié)論：高保真酶介導(dǎo)的突變敏感性分子開關(guān)可用于已知基因突變的檢測(cè)，，除可用于檢測(cè)某些遺傳性突變外，還可以檢測(cè)耐藥基因突變。高保真DNA聚合酶偶聯(lián)硫化修飾引物構(gòu)成的突變敏感性分子開關(guān)能夠快速篩查HIV-1七種突變，該技術(shù)在HIV-1耐藥突變檢測(cè)具有較大的潛在應(yīng)用價(jià)值，可用來(lái)指導(dǎo)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(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】：2013
【分類號(hào)】：R440;R512.91
本文編號(hào)：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(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】：2013
【分類號(hào)】：R440;R512.91
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