新型HIV-1基因型耐药检测方法及耐药准种动态的变化研究

发布时间：2018-05-06 03:29

本文选题：基因型耐药突变检测 + 干血斑　；参考：《中国疾病预防控制中心》2015年博士论文

【摘要】：研究目的和意义:目前控制HIV-1流行和传播最为有效的方法是高效抗逆转录病毒治疗,由于HIV病毒高度易变,伴随着患者服药依从性不佳等诸多因素,HIV-1耐药相关问题成为影响抗病毒治疗效果的主要因素。高效、敏感的HIV-1耐药突变的相关检测至关重要。HIV基因型耐药突变的检测方法较表现型的检测方法有诸多优势,但是目前常规应用的HIV-1基因型耐药突变检测方法有一定的局限性,如:血液样本直接运输受到生物安全和运输条件等诸多限制;在检测过程中有较多的人为因素;常规测序方法不能检测出低频耐药突变等。建立新型的HIV-1基因型耐药检测方法十分必要,同时研究在长期高效抗逆转录病毒治疗(HAART)下的HIV-1耐药准种的变化情况对治疗效果的影响有重要意义。研究方法:1.基于干血斑(Dried Blood Spot,DBS)样本完善HIV-1基因型耐药突变的检测方法,并对所建立方法的敏感性,准确性和可重复性进行系统评价。2.基于液态悬浮芯片技术建立HIV-1基因型耐药突变的检测方法,使用临床样本对检测方法进行验证。3.基于Illumina深度测序技术建立HIV-1基因型耐药突变的检测和分析方法,并利用此方法研究长期HAART作用下HIV-1感染者体内耐药准种的变化情况。研究结果:1.针对DBS样本完善HIV-1基因型耐药突变的检测方法:对于病毒载量介于(1000-6000 copies/ml)的79份DBS样本的检测敏感度为96.2%(76/79),核苷酸序列一致率分别为99.7±0.34和99.6±0.25%,Kappa统计值分别为0.972和0.963。比较64份DBS和血浆配对样本的基因型耐药突变位点的检测结果,该方法的敏感性和特异性分别为 99.49%(95%,97.4-99.8%)和 99.8%(95%,99.7-99.99%),有15个基因型耐药突变位点显示不一致,主要(83.3%,8/15)是由混合碱基导致。2.基于液态悬浮芯片技术建立了能检测HIV-1 B'亚型主要耐药突变位点的方法,较AS-PCR等方法,该方法可一次性检测多个耐药突变位点,结合我国抗病毒治疗药物使用情况,建立了涵盖我国NRTIs和NNRTIs主要突变位点的检测方法,并使用野生型质粒评价M41L,M184V探针的敏感度分别为25%,而K103N,V106A,K70R,K219Q和Q151M突变型检测探针的检测敏感度分别均为:12.5%,12.5%,12.5%,12.5%,12.5%;对我国己发生耐药的临床样本的检出率分别为:M41L 为 100%(16/16),M184V 为 85.7%(30/35),K103N 为 89.2%(25/28),V106A 为 83.3%(10/11),K70R 为 100%(14/14),K219Q 为 92.5%(25/27)及Q151M 为 87.5%(7/8)。3.基于Illumina深度测序技术建立了能检测出1%以上劣势耐药突变的检测方法和基于Geneious(?)软件的数据分析流程体系。该方法均能检出Sanger测序方法检出的耐药突变位点,若该耐药突变是由混合碱基导致,则深度测序方法可反应出突变型准种所占比例。在长期HAAT作用下观察到一系列HIV-1耐药准种的动态变化:1.停药后6-10个月检出低频耐药位点。尤其是大量较稳定的NNRTIs低频耐药突变位点,相关准种所占比例为1.8-17.4%,特别是K103N(4.3%-17.4%)低频位点在停药间长期存在,但是使用常规Sanger测序的HIV-1基因型耐药突变位点未能检出。2.在长期同一药物组方作用下,可见含有对该药物产生耐药的准种随治疗时间有明显的上升过程(1.1-97.1%)。3.在治疗组方中更换单药3TC时,可观察到含有M184V耐药准种从3.4%逐渐增加到97.4%的过程。4.部分最终死亡的HIV-1感染者在开始抗病毒治疗3-12个月即可见大量低频(1-15.6%)耐药突变,但是使用常规Sanger测序的HIV-1基因型耐药突变位点未能检出。5.更换二线抗病毒治疗方案之后仍产生NRTIs和NNRTIs耐药突变的患者,但未检出蛋白酶类相关低频耐药突变位点。结论:本研究以HIV-1基因型耐药突变检测方法为切入点,从样本类型的选择,高通量快速检测及对微量的劣势耐药突变的需求三个角度出发,建立了三种新型的HIV-1基因型耐药检测方法,可分别应用于不同检测目的。病毒载量小于6000拷贝的干血斑样本在也可以达到与普通血浆标本同样的耐药检测结果;应用液相芯片技术可以快速实现对影响耐药程度的主要位点筛查,可及时为患者治疗方案的选择服务;开发了方便学习使用的基于Geneious(?)软件的深度测序数据分析方法和流程。建立了易掌握可推广的应用深度测序方法进行耐药检测和分析研究的方法。在停止抗病毒治疗期间样本中,检出常规测序方法未检出的NNRTIs劣势耐药突变位点,可观察到不同的耐药准种在同一药物组方治疗下逐渐增长过程,部分死亡患者在治疗早期便检出低频耐药突变。
[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.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R512.91

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