基于逆转录环介导等温扩增技术的高灵敏度HIV-1 RNA检测

发布时间：2018-02-04 11:54

本文关键词： 逆转录环介导等温扩增（RT-LAMP）人类免疫缺陷病毒I型（HIV-1）现场检测羟基萘酚蓝（HNB）定量检测　出处：《华南理工大学》2013年硕士论文　论文类型：学位论文

【摘要】：艾滋病是一种致死性传染病，由人类免疫缺陷病毒HIV引起，该病毒分为HIV-1和HIV-2两型，前者是导致艾滋病的主要原因。中国是目前世界上艾滋病疫情发展较快的国家之一，且目前世界上没有可有效预防该疾病的疫苗和治愈艾滋病的特效药物。因此，HIV的检测成为控制和预防艾滋病的关键环节，是尽早发现HIV感染者、掌握疫情和病程、指导抗病毒治疗和疗效判定的有效手段。我们需要一种检测方法，它既有很高的敏感性、特异性，又要缩短窗口期、操作简便、快速和经济，从而增加检测可及性，能在现场检测即时出结果，以提升目前的检测水平和扩大应用范围。本论文利用一种新型、快速的核酸检测方法——逆转录环介导等温扩增技术建立了基于HNB颜色判定的RT-LAMP高灵敏度定性检测HIV-1RNA体系和基于RT-LAMP的快速定量HIV-1RNA体系。针对HIV-1gag保守基因设计RT-LAMP引物，使之能特异高效的检测出病毒基因。在RT-LAMP定性检测中，本研究将HNB与RT-LAMP技术结合，使得反应结果可通过肉眼直接观测，，脱离了对仪器的依赖，对该方法的灵敏度、特异性及临床样本检测等一系列性能进行了评估。在病毒载量定量实验中，本实验将RT-LAMP与HIV-1RNA的定量相结合，考察了该方法的定量性能（包括标准曲线的建立、最低检测限、实验再现性、特异性等），并通过与qRT-PCR对临床样本检测能力的一致性分析，评估了本方法在临床应用上的可行性。本研究结果表明，利用可视化的RT-LAMP体系检测HIV-1RNA，其引物灵敏度可达到5copies RNA，与其他血源性、同致病机制病毒（HTLV-1、HIV-2和HCV）无交叉反应，引物特异性良好；加入HNB的RT-LAMP体系能够正确的反映扩增的阴阳性，与琼脂糖凝胶电泳及实时浊度监测结果一致，且120μM的HNB不会降低引物的灵敏度；通过对95份临床样本分别进行RT-LAMP和qRT-PCR发现，该RT-LAMP体系的敏感性为95.29%，特异性为100%，假阳性率为0%，假阴性率为4.71%，功效率为95.79%，阳性预示值为100%，阴性预示值为71.43%，通过Kappa检验得知Kappa值=0.810，说明两种检测方法的一致性较好。利用RT-LAMP进行HIV-1RNA定量检测时发现，该方法的定量范围为2.5×102~1.0×107copies RNA，R2为0.991，最低检测限为196copies RNA，实验再现性良好，特异性为100%；对42份临床样本进行RT-LAMP定量和qRT-PCR，对其结果进行Bland-Altman分析，发现两种定量方法一致性为95.24%，p值＜0.05，说明方法间一致性良好。通过本论文的研究证明，RT-LAMP是一种快速、高效的HIV-1核酸现场检测手段，同时又具备HIV-1RNA的定量性能，有助于提高我国HIV检测技术水平，从而有效控制HIV的传播，辅助艾滋病病程监测与临床用药指导，具有极其广阔的应用前景。
[Abstract]:AIDS is a fatal infectious disease caused by the human immunodeficiency virus (HIV), the virus is divided into two types of HIV-1 and HIV-2. The former is the main cause of AIDS. China is one of the countries with rapid development of the AIDS epidemic in the world. At present, there is no effective vaccine to prevent the disease and a special drug to cure AIDS in the world. Therefore, the detection of HIV becomes the key link to control and prevent AIDS, is to find HIV infection as soon as possible. We need a detection method which has high sensitivity, specificity, short window period, simple operation, fast and economical. Thus, the detection accessibility can be increased, and the immediate results can be obtained in the field, so as to improve the current detection level and expand the scope of application. This paper uses a new type. A rapid nucleic acid detection method, reverse transcription-ring mediated isothermal amplification technique, was developed to establish a high sensitivity qualitative detection HIV-1RNA system for RT-LAMP based on HNB color determination and a HIV-1RNA system based on RT-LAM. The RT-LAMP primers were designed for the conserved gene of HIV-1gag. In the RT-LAMP qualitative detection, the HNB and RT-LAMP technology are combined to make the reaction results can be directly observed by the naked eye. A series of properties such as sensitivity, specificity and clinical sample detection of the method were evaluated without the dependence of the instrument. In this experiment, the quantitative properties of RT-LAMP and HIV-1RNA were investigated, including the establishment of standard curve, the minimum detection limit, the reproducibility and specificity of the experiment. The feasibility of this method in clinical application was evaluated by consistency analysis with qRT-PCR on clinical sample detection ability. The results showed that the primer sensitivity of HIV-1 RNA detected by visualized RT-LAMP system could reach 5 copies RNAs, and the primer sensitivity was similar to that of other blood sources. There was no cross reaction with HTLV-1 and HIV-2 and HCV, and the primer specificity was good. The RT-LAMP system added with HNB could correctly reflect the yin-yang character of amplification, which was consistent with the results of agarose gel electrophoresis and real-time turbidimetric monitoring, and the sensitivity of primers could not be reduced by 120 渭 M HNB. The RT-LAMP and qRT-PCR of 95 clinical samples showed that the sensitivity and specificity of the RT-LAMP system were 95.2929 and 100% respectively. False positive rate was 0, false negative rate was 4.71, efficacy rate was 95.79, positive predictive value was 100, negative predictive value was 71.43%. The results of Kappa test showed that the Kappa value was 0.810, which indicated that the consistency of the two detection methods was good. When using RT-LAMP to detect HIV-1RNA quantitatively, it was found. The quantitative range of the method was 2.5 脳 10 ~ (2) 脳 10 ~ (7) copies RNA R2 was 0.991.The minimum detection limit was 196 copies RNA. The reproducibility of the experiment was good and the specificity was 100. RT-LAMP quantitative analysis and qRT-PCR were performed in 42 clinical samples. The results of Bland-Altman analysis showed that the consistency of the two quantitative methods was 95.24%. P < 0.05, indicating good consistency between the methods. It is proved that RT-LAMP is a fast and efficient method for the detection of HIV-1 nucleic acid in situ and has the quantitative properties of HIV-1RNA. It is helpful to improve the technical level of HIV detection in China, so as to effectively control the spread of HIV, to assist in the monitoring of the course of AIDS and the guidance of clinical drug use, and has a very broad application prospect.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.91

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