本文選題：基因型耐藥突變檢測 + 干血斑　； 參考：《中國疾病預(yù)防控制中心》2015年博士論文

【摘要】：研究目的和意義:目前控制HIV-1流行和傳播最為有效的方法是高效抗逆轉(zhuǎn)錄病毒治療,由于HIV病毒高度易變,伴隨著患者服藥依從性不佳等諸多因素,HIV-1耐藥相關(guān)問題成為影響抗病毒治療效果的主要因素。高效、敏感的HIV-1耐藥突變的相關(guān)檢測至關(guān)重要。HIV基因型耐藥突變的檢測方法較表現(xiàn)型的檢測方法有諸多優(yōu)勢,但是目前常規(guī)應(yīng)用的HIV-1基因型耐藥突變檢測方法有一定的局限性,如:血液樣本直接運輸受到生物安全和運輸條件等諸多限制;在檢測過程中有較多的人為因素;常規(guī)測序方法不能檢測出低頻耐藥突變等。建立新型的HIV-1基因型耐藥檢測方法十分必要,同時研究在長期高效抗逆轉(zhuǎn)錄病毒治療(HAART)下的HIV-1耐藥準(zhǔn)種的變化情況對治療效果的影響有重要意義。研究方法:1.基于干血斑(Dried Blood Spot,DBS)樣本完善HIV-1基因型耐藥突變的檢測方法,并對所建立方法的敏感性,準(zhǔn)確性和可重復(fù)性進行系統(tǒng)評價。2.基于液態(tài)懸浮芯片技術(shù)建立HIV-1基因型耐藥突變的檢測方法,使用臨床樣本對檢測方法進行驗證。3.基于Illumina深度測序技術(shù)建立HIV-1基因型耐藥突變的檢測和分析方法,并利用此方法研究長期HAART作用下HIV-1感染者體內(nèi)耐藥準(zhǔn)種的變化情況。研究結(jié)果:1.針對DBS樣本完善HIV-1基因型耐藥突變的檢測方法:對于病毒載量介于(1000-6000 copies/ml)的79份DBS樣本的檢測敏感度為96.2%(76/79),核苷酸序列一致率分別為99.7±0.34和99.6±0.25%,Kappa統(tǒng)計值分別為0.972和0.963。比較64份DBS和血漿配對樣本的基因型耐藥突變位點的檢測結(jié)果,該方法的敏感性和特異性分別為 99.49%(95%,97.4-99.8%)和 99.8%(95%,99.7-99.99%),有15個基因型耐藥突變位點顯示不一致,主要(83.3%,8/15)是由混合堿基導(dǎo)致。2.基于液態(tài)懸浮芯片技術(shù)建立了能檢測HIV-1 B'亞型主要耐藥突變位點的方法,較AS-PCR等方法,該方法可一次性檢測多個耐藥突變位點,結(jié)合我國抗病毒治療藥物使用情況,建立了涵蓋我國NRTIs和NNRTIs主要突變位點的檢測方法,并使用野生型質(zhì)粒評價M41L,M184V探針的敏感度分別為25%,而K103N,V106A,K70R,K219Q和Q151M突變型檢測探針的檢測敏感度分別均為:12.5%,12.5%,12.5%,12.5%,12.5%;對我國己發(fā)生耐藥的臨床樣本的檢出率分別為:M41L 為 100%(16/16),M184V 為 85.7%(30/35),K103N 為 89.2%(25/28),V106A 為 83.3%(10/11),K70R 為 100%(14/14),K219Q 為 92.5%(25/27)及Q151M 為 87.5%(7/8)。3.基于Illumina深度測序技術(shù)建立了能檢測出1%以上劣勢耐藥突變的檢測方法和基于Geneious(?)軟件的數(shù)據(jù)分析流程體系。該方法均能檢出Sanger測序方法檢出的耐藥突變位點,若該耐藥突變是由混合堿基導(dǎo)致,則深度測序方法可反應(yīng)出突變型準(zhǔn)種所占比例。在長期HAAT作用下觀察到一系列HIV-1耐藥準(zhǔn)種的動態(tài)變化:1.停藥后6-10個月檢出低頻耐藥位點。尤其是大量較穩(wěn)定的NNRTIs低頻耐藥突變位點,相關(guān)準(zhǔn)種所占比例為1.8-17.4%,特別是K103N(4.3%-17.4%)低頻位點在停藥間長期存在,但是使用常規(guī)Sanger測序的HIV-1基因型耐藥突變位點未能檢出。2.在長期同一藥物組方作用下,可見含有對該藥物產(chǎn)生耐藥的準(zhǔn)種隨治療時間有明顯的上升過程(1.1-97.1%)。3.在治療組方中更換單藥3TC時,可觀察到含有M184V耐藥準(zhǔn)種從3.4%逐漸增加到97.4%的過程。4.部分最終死亡的HIV-1感染者在開始抗病毒治療3-12個月即可見大量低頻(1-15.6%)耐藥突變,但是使用常規(guī)Sanger測序的HIV-1基因型耐藥突變位點未能檢出。5.更換二線抗病毒治療方案之后仍產(chǎn)生NRTIs和NNRTIs耐藥突變的患者,但未檢出蛋白酶類相關(guān)低頻耐藥突變位點。結(jié)論:本研究以HIV-1基因型耐藥突變檢測方法為切入點,從樣本類型的選擇,高通量快速檢測及對微量的劣勢耐藥突變的需求三個角度出發(fā),建立了三種新型的HIV-1基因型耐藥檢測方法,可分別應(yīng)用于不同檢測目的。病毒載量小于6000拷貝的干血斑樣本在也可以達到與普通血漿標(biāo)本同樣的耐藥檢測結(jié)果;應(yīng)用液相芯片技術(shù)可以快速實現(xiàn)對影響耐藥程度的主要位點篩查,可及時為患者治療方案的選擇服務(wù);開發(fā)了方便學(xué)習(xí)使用的基于Geneious(?)軟件的深度測序數(shù)據(jù)分析方法和流程。建立了易掌握可推廣的應(yīng)用深度測序方法進行耐藥檢測和分析研究的方法。在停止抗病毒治療期間樣本中,檢出常規(guī)測序方法未檢出的NNRTIs劣勢耐藥突變位點,可觀察到不同的耐藥準(zhǔn)種在同一藥物組方治療下逐漸增長過程,部分死亡患者在治療早期便檢出低頻耐藥突變。
[Abstract]:Objective and significance: the most effective method to control the epidemic and transmission of HIV-1 is the effective antiretroviral therapy. Due to the high variability of the HIV virus and the poor compliance with the patient, the HIV-1 resistance related problems are the main factors affecting the effect of antiviral therapy. The highly efficient, sensitive HIV-1 resistance mutation The detection methods of.HIV genotypic resistance mutation are more important than those of the phenotype detection methods. However, there are some limitations in the conventional HIV-1 genotypic resistance mutation detection methods, such as: the direct transportation of blood samples is limited by biological safety and transportation conditions, and there are more in the detection process. Human factors; the conventional sequencing method can not detect the low frequency resistance mutation. It is necessary to establish a new type of HIV-1 genotypic resistance detection method. Meanwhile, the study on the changes of the HIV-1 resistant quasi species under the long-term high performance antiretroviral therapy (HAART) is of great significance to the effect of the treatment. 1. based on the dry blood spot (D Ried Blood Spot, DBS) samples improved the detection method of HIV-1 genotypic resistance mutation, and systematically evaluated the sensitivity, accuracy and repeatability of the established methods..2. based on liquid suspension chip technology to establish the detection method of HIV-1 genotypic resistance mutation, using clinical samples to verify the detection method based on the Illumina depth of.3.. The method of detection and analysis of HIV-1 genotypic resistance mutation was established by sequencing technology, and the change of drug resistant quasi species in HIV-1 infected persons under the action of long term HAART was studied by this method. 1. the results of the study were as follows: the detection method of HIV-1 genotypic resistance mutation for DBS samples: 79 DBS samples (1000-6000 copies/ml) of the disease drug load. The sensitivity of the detection was 96.2% (76/79), the nucleotide sequences were 99.7 + 0.34 and 99.6 + 0.25% respectively. The Kappa statistical values were 0.972 and 0.963. compared to 64 DBS and plasma paired samples. The sensitivity and specificity of this method were 99.49% (95%, 97.4-99.8%) and 99.8% (95%, 99.7-99, respectively). .99%), 15 genotypic mutant loci are inconsistent. The main (83.3%, 8/15) is a method to detect the main resistance mutation loci of the HIV-1 B'subtype by the liquid suspension chip technology, which is based on the mixture base, which is based on the liquid suspension chip technology. The method can be used to detect multiple resistance mutation sites and combine with our antiviral drugs in one time. This method can be used to detect multiple resistance mutations at one time. The detection methods covering the main mutation sites of NRTIs and NNRTIs in China were established, and the sensitivity of the M184V probe was 25%, respectively, using the wild type plasmid, while the sensitivity of K103N, V106A, K70R, K219Q and Q151M mutagenesis probes were 12.5%, 12.5%, 12.5%, 12.5%, 12.5%, respectively. The detection rates of clinical samples were as follows: M41L was 100% (16/16), M184V was 85.7% (30/35), K103N was 89.2% (25/28), V106A was 83.3% (10/11), K70R was 100% (14/14), K219Q was 92.5% (25/27) and 87.5% (87.5%) built a detection method and based on the detection of more than 1% disadvantaged mutations based on the depth sequencing technology. Eious (?) software data analysis process system. This method can detect the resistance mutation loci detected by Sanger sequencing. If the resistance mutation is caused by mixed bases, then the depth sequencing method can reflect the proportion of the mutant quasispecies. The dynamic changes of a series of HIV-1 resistant quasispecies are observed under the long-term HAAT effect: 1. after the drug withdrawal, 6- The low frequency resistance loci were detected in 10 months, especially a large number of more stable NNRTIs low-frequency resistance mutation sites. The proportion of the related quasi species was 1.8-17.4%, especially the low frequency loci of K103N (4.3%-17.4%) existed for a long time in the stopping room, but the HIV-1 genotypic mutant loci of the conventional Sanger sequencing failed to detect.2. in the long-term same drug group. Under the action, it can be seen that the quasispecies which have resistance to the drug have an obvious increase in the time of treatment (1.1-97.1%).3., when the single drug 3TC is replaced in the treatment group, the M184V resistant quasispecies from 3.4% to 97.4% have been gradually increased to the.4. part of the final death of the HIV-1 infected persons at the beginning of the antiviral treatment for 3-12 months. A large number of low frequency (1-15.6%) resistance mutations, but the use of conventional Sanger sequenced HIV-1 genotype resistance mutation sites failed to detect the patients with NRTIs and NNRTIs resistant mutations after the.5. replacement of the second line antiviral therapy, but the protease related low-frequency resistance mutation sites were not detected. Conclusion: This study was based on the mutation of the HIV-1 genotype. The detection method is the breakthrough point. From the selection of sample type, high throughput rapid detection and the demand for minor drug resistance mutation, three new types of HIV-1 genotypic resistance detection methods are established, which can be applied to different detection purposes. The dry blood samples with the viral load less than 6000 copies can also be achieved with the common type. The same results of resistance detection in the plasma specimens; the application of liquid chip technology can quickly screen the major sites affecting the degree of resistance, and can serve the patient's treatment options in a timely manner, and develop a method and process for the analysis of the depth sequencing based on Geneious (?) software, which is easy to learn and used. The method of deep sequencing was used to detect and analyze the drug resistance. In the sample of stopping antiviral therapy, the NNRTIs disadvantaged mutant loci were detected undetected by the conventional sequencing method. The gradual growth process of different drug resistant quasispecies was observed under the treatment of the same drug group, and some dead patients were detected in the early stage of treatment. Low frequency resistance mutation.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R512.91

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本文編號：1850598