三种PCR技术在HIV诊断及疗效监测中的应用研究

发布时间：2018-05-08 02:08

本文选题：1型艾滋病病毒 + 核酸定性检测　；参考：《中国疾病预防控制中心》2017年硕士论文

【摘要】：目前常用的核酸定性检测技术包括:实验室自建的In-house方法、Abbott M2000HIV-1 RNA/DNA以及AptimaHIV-1RNA核酸定性检测。常用的核酸定量检测技术包括罗氏诊断的Cobas Amplipre/Cobas TaqMan HTV-1 Testv 2.0、雅培公司的RealTime HIV-1、梅里埃公司的 Nuclisens EasyQ HIV-1 v2.0 和西门子公司的 Versant HIV-1 RNA 3.0 assay(b-DNA)。随着分子生物学技术的发展,更灵敏的核酸检测方法应运而生,包括数字PCR技术等。本研究采用Nuclisens EasyQ HIV-1核酸定量检测、PCR 荧光探针法和数字 PCR(digita1 polymerase chain reaction,dPCR)对 HIV诊断及治疗后进行了系统研究,主要包括以下三个部分:第一部分核酸定量检测采用梅里埃公司的Nuclisens EasyQ HIV-1 v2.0,直接检测血浆中HIV的RNA量。全面地分析核酸定性以及定量检测的结果,深入地探讨在实验室中,核酸定量检测用于诊断HIV的可行性以及可操作性。第二部分采用PCR-荧光探针法检测HIV患者治疗后PBMCs内HIV-1前病毒DNA的水平,探索在血浆HIV-1 RNA低于检测下限时,血细胞HIV-1前病毒作为判断抗病毒治疗疗效以及病情进展预测指标的价值,为进一步探索清除病毒的新治疗措施奠定基础。第三部分采用ddPCR、qPCR对不同月龄的HIV感染新生儿进行检测,比较两种方法的灵敏度和特异度;并评价ddPCR、qPCR两种检测方法的一致性,为制定我国的HIV暴露婴儿的实验室诊断检测策略做参考依据。第一部分核酸定量检测应用于HIV-1感染诊断的研究研究背景艾滋病(Acquired immune deficiency syndrome,AIDS)的病原体是人类免疫缺陷病毒(Human Immunodeficiency Virus,HIV),2017 年 2 月,全国(不含港澳台)共报告法定传染病485649例,死亡1409例,其中AIDS死亡人数1097例(77.9%)。将HIV及时地发现并且及早期地诊断出对艾滋感染者的治疗非常紧要,重要的是它能够有效地降低患者的死亡率、并且能够将其生存的质量提高。艾滋病目前常用的检测手段主要有抗体检测和病毒检测,病毒检测主要包括p24抗原检测、细胞培养(病毒分离)以及核酸检测。抗体检测通常是在感染后3-8周才能检测出来,而核酸检测能够在感染后2周内检测出来,从而缩短窗口期。核酸检测的好处有两个即敏感性以及特异性较高,因此能够作为艾滋早期诊断的重要手段。根据《全国艾滋病检测技术规范》(2015年)修订版中的规定:检测策略补充试验包括抗体确证以及核酸试验,将核酸检测提到了艾滋病检测诊断的关键地位。定量检测能够将外周血中的HIV病毒含量测出,比定性检测更加便捷和敏感。本研究中核酸定性检测采用中国疾病预防控制中心性病艾滋病中心参比实验室自建的In-house方法,应用人类所有细胞均含有的β-actin基因作内参,当先扩增出β-actin片段后再根据巢式的方法用pol区、gag区、env区相对应的引物来扩增HIV特异性的基因片段,最终结果根据扩增产物来判定。核酸定量检测则采用梅里埃公司的Nuclisens EasyQ HIV-1 v2.0,直接检测血浆中HIV的RNA量。全面地分析核酸定性以及定量检测的结果,深入地探讨在实验室中,核酸定量检测用于诊断HIV的可行性以及可操作性。目的探讨核酸定量检测在1型艾滋病病毒(HIV-1)实验室感染诊断中的应用。对象和方法选取云南省德宏州216例样本,包括136例HIV-1阳性母亲所生活产婴儿和80例HIV-1阳性成人,分别用HIV-1核酸定性和定量方法进行检测,综合对比分析2种方法的检测结果。结果1.216例样本的核酸定性和定量检测试验一致性极好,53例样本(50例成人样本和3例婴儿样本)核酸阳性,血浆病毒载量5000CPs/mL,随访50例成人样本的蛋白印迹试验(WB)确证均为HIV-1抗体阳性;2.163例阴性样本(30例成人样本和133例婴儿样本)核酸阴性,血浆病毒载量均低于检测下限。结论当血浆病毒载量5000CPs/mL,核酸定量检测试验对于HIV-1感染的阳性样本是一种有效的实验室诊断方法。第二部分PCR-荧光探针法在成人HIV/AIDS接受长期HAART治疗后的应用研究研究背景人体感染HIV后可整合在宿主基因上,从而使病毒潜伏在淋巴细胞,脑细胞以及消化道淋巴组织等组织器官中,形成了病毒储存库,高效抗逆转录病毒治疗不能彻底清除人体内的病毒。病毒潜伏或潜伏病毒即病毒在被感染的细胞中暂不复制,可逃避宿主的免疫清除,但在一定的条件下可复制病毒。HAART能够很明显地降低艾滋病的病死率,也能够抑制病毒的有效复制,同时不仅将HIV/AIDS血浆中的病毒载量降低至目前现存的检测方法的最低检测水平以下,而且还能升高CD4+T细胞数量,很好地重新构建人体的免疫功能。但是,至今为止尚不能彻底治愈HIV,可能是因为人体内会持续地存在病毒储存库。本研究采用PCR-荧光探针法检测HIV患者治疗后PBMCs内HIV-1前病毒DNA的水平,拟探索在血浆HIV-1 RNA低于检测下限时,将PBMCs细胞内HIV-1前病毒水平作为判断抗病毒治疗疗效以及病情进展预测指标的价值,为进一步探索清除病毒的新治疗措施奠定基础。目的探讨PCR-荧光探针法检测成人HIV/AIDS接受高效抗逆转录病毒治疗(HAART)后HIV-1病毒储存库的改变情况及病毒储存库与治疗效果的关系。对象和方法对91例规范接受HAART治疗5-10年的HIV-1患者采集血标本进行横断面研究;通过分支DNA(bDNA)法检测血浆HIV-1RNA病毒载量;实验室自建的In-house巢式PCR、PCR-荧光探针法检测外周血HIV-IDNA水平;流式细胞术检测外周血CD4+T细胞数量;对数据进行统计学处理。结果1.91例接受5-10年HAART治疗的HIV-1患者外周血HIV-1RNA均低于检测下限(50拷贝/ml);2.细胞中HIV-1前病毒DNA的平均含量为1.77±0.76(log10拷贝/106个细胞);3.实验室自建的In-house巢式PCR法检测细胞中HIV-1 DNA阴性者比例为39/91,不确定者比例为52/91;4.CD4+T细胞平均数量为479.56±188.11个/μl。结论PCR-荧光探针法检测经过长期HAART治疗(5-10年)成人患者的病毒储存库更加灵敏,储存库的检测对于抗病毒治疗疗效的判断以及病情进展的预测都有重要的意义。第三部分数字PCR和荧光定量PCR用于HIV婴儿早期诊断的研究研究背景HIV-l DNA可以整合到宿主DNA中,即为前病毒。而细胞内总的HIVDNA是测量储存库大小的指标。HIVDNA对于HIV的诊断、疗效监测具有很重要的意义。目前HIV DNA定量检测主要采用实时荧光定量PCR(real—time quantitation PCR,qPCR),其依赖的定量基础循环阈值(cycle threshold,Ct)受扩增效率的影响很大,故qPCR只能相对定量。微滴式数字PCR(droplet digital polymerase chain reaction,ddPCR)是一种核酸检测和绝对定量的新方法,将样品通过微滴发生器进行微滴化处理,形成数以万计的油包水小液滴,每个液滴中含有不超过一个DNA分子,每个DNA分子均作为单独的PCR反应体系,经PCR扩增后与荧光标记探针杂交,用微滴检测器对每个反应进行检测,实现对单分子核酸的绝对定量。ddPCR技术无需建立标准曲线,且具有较高的灵敏度,但因其价格较高,所以国内还没有其在HIV定量检测方面的研究。本研究拟采用ddPCR、qPCR对不同月龄的HIV感染新生儿进行检测,比较两种方法的灵敏度和特异度,并评价ddPCR、qPCR两种检测方法的一致性;为制定我国的HIV暴露婴儿的实验室诊断检测策略做参考依据。目的比较ddPCR、qPCR对不同月龄的HIV感染新生儿检测的灵敏度与特异度,并评价ddPCR、qPCR两种检测方法的一致性。对象和方法选取2014-2016年云南、四川、新疆、重庆4省的HIV-1阳性母亲所生活产婴儿116例不同时间点干血斑(DBS)样本,根据《全国艾滋病检测技术规范》2015年修订版,本研究的金标准为连续两次核酸定性检测为阳性结果即判定为阳性。即116例婴儿干血斑样本中,有54例阳性样本,62例阴性样本。结果1.116例婴儿样本,qPCR的灵敏度为63.0%,特异度为91.9%;ddPCR的灵敏度为59.2%,特异度为75.8%;qPCR与ddPCR检测一致率为73.3%,Kappa=0.391;2.对于产后24h内的样本而言,qPCR的灵敏度为36.3%,特异度为80.0%;ddPCR的灵敏度为27.2%,特异度为73.3%;qPCR与ddPCR检测一致率为53.8%,Kappa=-0.173;3.对于产后4w内的样本,qPCR的灵敏度为50.0%,特异度为100.0%;ddPCR的灵敏度为57.1%,特异度为92.9%。qPCR与ddPCR检测一致率为78.6%,Kappa=0.478;4.对于产后6W样本,qPCR的灵敏度为78.6%,特异度为100%;ddPCR的灵敏度为78.6%,特异度为77.8%。qPCR与ddPCR检测一致率为87.5%,Kappa=0.745;5.对于产后3m样本qPCR的灵敏度为80.0%,特异度为86.7%;ddPCR的灵敏度为66.7%,特异度为60.0%。qPCR与ddPCR检测一致率为66.7%,Kappa=0.336。结论ddPCR与qPCR两种检测方法的灵敏度与特异度均不高,且两种检测方法一致性较差,暂不能作为婴儿HIV早期诊断检测的参考依据。
[Abstract]:Currently, the commonly used nucleic acid detection techniques include the In-house method built by the laboratory, the Abbott M2000HIV-1 RNA/DNA and the qualitative detection of AptimaHIV-1RNA nucleic acid. The commonly used nucleic acid quantitative detection techniques include Roche's Cobas Amplipre/Cobas TaqMan HTV-1 Testv 2, the RealTime HIV-1 of the Abbott Company, and the mill of the company. EasyQ HIV-1 v2.0 and SIEMENS's Versant HIV-1 RNA 3 assay (b-DNA). With the development of molecular biology technology, more sensitive nucleic acid detection methods have come into being, including digital PCR technology, etc. This study uses Nuclisens EasyQ HIV-1 nucleic acid quantitative detection. On, dPCR) carried out a systematic study on the diagnosis and treatment of HIV, including the following three parts: the first part of the quantitative detection of nucleic acid, using the Nuclisens EasyQ HIV-1 v2.0 of the company, to determine the RNA amount of HIV in the plasma directly. The results of nucleic acid determination and quantitative detection are analyzed comprehensively. The nucleic acid quantification in the laboratory is deeply discussed. The feasibility and maneuverability of detecting HIV were detected. The second part used PCR- fluorescence probe to detect the level of HIV-1 virus DNA in PBMCs after HIV treatment, and explored the value of the pre HIV-1 virus of blood cells as the value of judging the curative effect of antiviral treatment and the predictor of the progression of the disease when the plasma HIV-1 RNA was lower than the detection limit. The third part uses ddPCR and qPCR to detect the HIV infected newborns with different months of age, compare the sensitivity and specificity of the two methods, and evaluate the consistency of the two methods of ddPCR and qPCR, and make reference to the formulation of laboratory diagnostic testing strategies for HIV exposed infants in China. The first part of the quantitative detection of nucleic acid is applied to the diagnosis of HIV-1 infection. The pathogen of Acquired immune deficiency syndrome (AIDS) is the human immunodeficiency virus (Human Immunodeficiency Virus, HIV). In February 2017, 485649 cases of statutory infectious diseases were reported in China (excluding Hong Kong, Macao and Taiwan), and 1409 cases died, of which A The number of IDS deaths is 1097 (77.9%). The timely detection and early diagnosis of HIV and the early diagnosis of AIDS infected people are very important. It is important that it can effectively reduce the death rate of the patient and improve the quality of its survival. The current detection hand of AIDS is mainly antibody detection and virus detection, virus detection. It mainly includes p24 antigen detection, cell culture (virus isolation) and nucleic acid detection. Antibody detection is usually detected at 3-8 weeks after infection, and nucleic acid detection can be detected within 2 weeks after infection, thus reducing the window period. The advantages of nucleic acid detection are two sensitivity and high specificity, so it can be used as early AIDS. An important means of phase diagnosis. According to the provisions of the National AIDS test specification (2015) revised edition: the test strategy supplementary test includes antibody confirmation and nucleic acid test, which refers to the key status of the detection and diagnosis of AIDS. Quantitative detection can detect the content of HIV virus in peripheral blood more than qualitative detection. It is convenient and sensitive. In this study, the nucleic acid qualitative detection used the In-house method built by the reference laboratory of the center for STD AIDS in the center for disease prevention and control of China. The beta -actin gene contained in all human cells was used as the internal reference, the beta -actin fragment was amplified first and then the corresponding primers in the pol region, gag region, and env region were used by the nested method. To amplify the specific gene fragment of HIV, the final result is determined according to the amplified product. The nucleic acid quantitative detection uses the Nuclisens EasyQ HIV-1 v2.0 of mrer company to directly detect the RNA quantity of HIV in the plasma. The results of nucleic acid qualitative and quantitative detection are analyzed comprehensively. The quantitative detection of nucleic acid in the laboratory is used to diagnose the nucleic acid in the laboratory. HIV's feasibility and maneuverability. Objective to explore the application of nucleic acid quantitative detection in the diagnosis of laboratory infection of type 1 AIDS virus (HIV-1). Objects and methods selected 216 samples from Dehong, Yunnan Province, including 136 cases of HIV-1 positive mothers living and 80 cases of HIV-1 positive adults, using HIV-1 nucleic acid qualitative and quantitative methods respectively. Results of the test, the results of the 2 methods were compared and analyzed. Results the consistency of the nucleic acid qualitative and quantitative test of 1.216 samples was excellent, 53 samples (50 adult samples and 3 infant samples) were positive for nucleic acid and plasma viral load 5000CPs/mL. The egg white blot test (WB) of 50 adult samples were confirmed to be HIV-1 antibody positive; 2.163 The negative samples (30 adult samples and 133 infant samples) were negative and the plasma viral load was lower than the lower detection limit. Conclusion when the plasma viral load is 5000CPs/mL, the nucleic acid quantitative detection test is an effective laboratory diagnosis method for the positive samples of HIV-1 infection. The second part of the PCR- fluorescence probe is accepted in adult HIV/AIDS. The application of HAART after treatment study background human infection HIV can be integrated on the host gene, which makes the virus latent in the tissues and organs such as lymphocytes, brain cells and digestive lymphatic tissues, and forms a virus store. High performance antiretroviral therapy can not clear the virus in the human body. Virus latent or latent disease Virus is not replicated in infected cells and escaping from the immune clearance of the host, but replicating virus.HAART under certain conditions can significantly reduce the mortality of AIDS and inhibit the effective replication of the virus, and not only reduce the viral load in the HIV/AIDS plasma to the most existing testing methods. Under low level of detection, the number of CD4+T cells can be increased and the immune function of the human body is rebuilt well. However, it is not possible to completely cure HIV so far. It may be because of the persistent presence of virus storage in the human body. This study used PCR- fluorescence probe to detect the level of HIV-1 pre virus DNA in PBMCs after the treatment of HIV patients. To explore the value of the level of HIV-1 pre virus in PBMCs cells as an indicator of the therapeutic effect of antiviral therapy and the predictor of progression of the disease in PBMCs cells when the plasma RNA is lower than the detection limit, and to explore the basis for further exploration of the new treatment measures for eliminating the virus. Objective to investigate the detection of adult HIV/AIDS by PCR- fluorescence probe for the acceptance of high performance antiretroviral drugs. The changes in the HIV-1 virus repository after treatment (HAART) and the relationship between the virus repository and the therapeutic effect. Objects and methods were used to conduct a cross-sectional study of the blood samples collected from 91 HIV-1 patients receiving HAART therapy for 5-10 years; the HIV-1RNA virus load in plasma was detected by the branch DNA (bDNA) method; the In-house nested PCR, PCR- fluore was built in the laboratory. The HIV-IDNA level of peripheral blood was detected by light probe, and the number of CD4+T cells in peripheral blood was detected by flow cytometry. The data were processed statistically. Results the peripheral blood HIV-1RNA of 1.91 cases of HIV-1 patients receiving 5-10 years of HAART treatment were lower than the lower detection limit (50 copies /ml), and the average content of HIV-1 pre virus DNA in 2. cells was 1.77 + 0.76 (log10 copy /106). 3. In-house nested PCR method was built in the laboratory to detect the proportion of HIV-1 DNA negative in the cells was 39/91, the ratio of the uncertain person was 52/91; the average number of 4.CD4+T cells was 479.56 + 188.11 / mu L. conclusion PCR- fluorescence probe was more sensitive and the storage library was detected after long-term HAART treatment (5-10 years). The judgment of the efficacy of antiviral therapy and the prediction of the progress of the disease are of great significance. Third parts of digital PCR and fluorescence quantitative PCR are used to study the early diagnosis of HIV infants. HIV-l DNA can be integrated into the host DNA, that is, the pre virus, and the total HIVDNA in the cell is a measure of the size of the repository.HIVDNA for HIV At present, the quantitative detection of HIV DNA mainly uses real-time quantitative PCR (real - time quantitation PCR, qPCR), and its dependent quantitative basic cycle threshold (cycle threshold, Ct) is greatly influenced by the amplification efficiency. Therefore, qPCR can only be relatively quantitative. Chain reaction, ddPCR) is a new method of nucleic acid detection and absolute quantification. The samples are microdripped by microdrop generator to form tens of thousands of small liquid droplets of oil water. Each droplet contains no more than one DNA molecule. Each DNA molecule is used as a separate PCR reaction system, and after PCR amplification with a fluorescent labeling probe Each reaction was detected by a micro drop detector. The absolute quantitative.DdPCR technology for single molecule nucleic acid was not required to establish a standard curve and had high sensitivity. But because of its high price, there was no study on quantitative detection of HIV in China. This study was intended to use ddPCR and qPCR for HIV infected newborns with different months of age. The sensitivity and specificity of the two methods were compared, and the consistency of the two methods of ddPCR and qPCR were evaluated and the reference basis for the laboratory diagnosis of HIV exposed infants in China. The purpose was to compare the sensitivity and specificity of ddPCR, qPCR to the detection of HIV infected newborns with different months of age, and to evaluate the two species of ddPCR and qPCR. Conformance of detection methods. Objects and methods selected 116 cases of HIV-1 positive mothers living in 4 provinces of Yunnan, Sichuan, Xinjiang, Chongqing and 116 cases of dry blood spots (DBS) at different time points. According to the National AIDS test specification >2015 revised edition, the gold standard of this study was the positive result of two consecutive tests of nucleic acid. There were 54 positive samples and 62 negative samples in the 116 samples of dry blood spots in 116 infants. The sensitivity of 1.116 infants was 63%, the specificity was 91.9%, the sensitivity of ddPCR was 59.2%, the specificity was 75.8%, and the coincidence rate of qPCR and ddPCR was 73.3%, Kappa=0.391; 2. for postpartum 24h samples, qPCR spirit. The sensitivity was 36.3%, the specificity was 80%, the sensitivity of ddPCR was 27.2%, the specificity was 73.3%, and the consistency between qPCR and ddPCR was 53.8%, Kappa=-0.173; 3. for postpartum 4W samples, the sensitivity of qPCR was 50%, the specificity was 100%; the sensitivity of ddPCR was 57.1%, and the specificity was 78.6%, Kappa=0.478; 4. for 92.9%.qPCR and ddPCR. After postpartum 6W samples, the sensitivity of qPCR was 78.6%, the specificity was 100%, the sensitivity of ddPCR was 78.6%, the coincidence rate of 77.8%.qPCR and ddPCR was 87.5%, Kappa=0.745; 5. for postpartum 3M samples, the sensitivity was 80%, the specificity was 86.7%, the sensitivity of ddPCR was 66.7%, the specificity of 60.0%.qPCR and ddPCR was 66.7%, Kappa was 66.7%, Kappa was 66.7%, Kappa was 66.7%, Kappa was 66.7%, Kappa was 66.7%, Kappa was 66.7%, Kappa was 66.7%, Kappa was 66.7%, Kappa was 66.7%, Kappa was 66.7%, Kappa =0.336. conclusion the sensitivity and specificity of the two detection methods of ddPCR and qPCR are not high, and the consistency of the two detection methods is poor, which can not be used as the reference basis for the early diagnosis and detection of infant HIV.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91

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