發(fā)布時間：2018-02-04 11:54

  本文關(guān)鍵詞： 逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫擴增（RT-LAMP） 人類免疫缺陷病毒I型（HIV-1） 現(xiàn)場檢測 羥基萘酚藍（HNB） 定量檢測　出處：《華南理工大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：艾滋病是一種致死性傳染病，由人類免疫缺陷病毒HIV引起，該病毒分為HIV-1和HIV-2兩型，前者是導(dǎo)致艾滋病的主要原因。中國是目前世界上艾滋病疫情發(fā)展較快的國家之一，且目前世界上沒有可有效預(yù)防該疾病的疫苗和治愈艾滋病的特效藥物。因此，HIV的檢測成為控制和預(yù)防艾滋病的關(guān)鍵環(huán)節(jié)，是盡早發(fā)現(xiàn)HIV感染者、掌握疫情和病程、指導(dǎo)抗病毒治療和療效判定的有效手段。我們需要一種檢測方法，它既有很高的敏感性、特異性，又要縮短窗口期、操作簡便、快速和經(jīng)濟，從而增加檢測可及性，能在現(xiàn)場檢測即時出結(jié)果，以提升目前的檢測水平和擴大應(yīng)用范圍。 本論文利用一種新型、快速的核酸檢測方法——逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫擴增技術(shù)建立了基于HNB顏色判定的RT-LAMP高靈敏度定性檢測HIV-1RNA體系和基于RT-LAMP的快速定量HIV-1RNA體系。針對HIV-1gag保守基因設(shè)計RT-LAMP引物，使之能特異高效的檢測出病毒基因。在RT-LAMP定性檢測中，本研究將HNB與RT-LAMP技術(shù)結(jié)合，使得反應(yīng)結(jié)果可通過肉眼直接觀測，，脫離了對儀器的依賴，對該方法的靈敏度、特異性及臨床樣本檢測等一系列性能進行了評估。在病毒載量定量實驗中，本實驗將RT-LAMP與HIV-1RNA的定量相結(jié)合，考察了該方法的定量性能（包括標準曲線的建立、最低檢測限、實驗再現(xiàn)性、特異性等），并通過與qRT-PCR對臨床樣本檢測能力的一致性分析，評估了本方法在臨床應(yīng)用上的可行性。 本研究結(jié)果表明，利用可視化的RT-LAMP體系檢測HIV-1RNA，其引物靈敏度可達到5copies RNA，與其他血源性、同致病機制病毒（HTLV-1、HIV-2和HCV）無交叉反應(yīng)，引物特異性良好；加入HNB的RT-LAMP體系能夠正確的反映擴增的陰陽性，與瓊脂糖凝膠電泳及實時濁度監(jiān)測結(jié)果一致，且120μM的HNB不會降低引物的靈敏度；通過對95份臨床樣本分別進行RT-LAMP和qRT-PCR發(fā)現(xiàn)，該RT-LAMP體系的敏感性為95.29%，特異性為100%，假陽性率為0%，假陰性率為4.71%，功效率為95.79%，陽性預(yù)示值為100%，陰性預(yù)示值為71.43%，通過Kappa檢驗得知Kappa值=0.810，說明兩種檢測方法的一致性較好。利用RT-LAMP進行HIV-1RNA定量檢測時發(fā)現(xiàn)，該方法的定量范圍為2.5×102~1.0×107copies RNA，R2為0.991，最低檢測限為196copies RNA，實驗再現(xiàn)性良好，特異性為100%；對42份臨床樣本進行RT-LAMP定量和qRT-PCR，對其結(jié)果進行Bland-Altman分析，發(fā)現(xiàn)兩種定量方法一致性為95.24%，p值＜0.05，說明方法間一致性良好。 通過本論文的研究證明，RT-LAMP是一種快速、高效的HIV-1核酸現(xiàn)場檢測手段，同時又具備HIV-1RNA的定量性能，有助于提高我國HIV檢測技術(shù)水平，從而有效控制HIV的傳播，輔助艾滋病病程監(jiān)測與臨床用藥指導(dǎo)，具有極其廣闊的應(yīng)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.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R512.91

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