RT-LAMP检测扎伊尔型埃博拉病毒的研究

发布时间：2018-06-21 14:59

本文选题：扎伊尔型埃博拉病毒 + 环介导恒温扩增技术　；参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：埃博拉病毒(Ebola virus)作为烈性病原体之一,在2014年的暴发流行引起了全球的关注。埃博拉病毒是最早出现在1976年暴发于西非埃博拉河地区两起疫情中,因此被命名为埃博拉。2014年,扎伊尔型埃博拉病毒病肆虐西非,其为自SARS以来最为严重的全球性公共卫生事件。WHO判定此次西非埃博拉疫情是自1976年埃博拉病毒被发现以来暴发的范围最广且最复杂的埃博拉疫情,这次疫情出现的感染人数和死亡人数超出了其它全部疫情的总和。埃博拉病毒传播速度极快,首先出现在西非的几内亚共和国,后传播至与其接壤的塞拉利昂和利比里亚,又由空中输入尼日利亚和美国,然后蔓延到马里和塞内加尔。此次疫情在西非、欧洲和美洲等9个国家共造成了29000余人感染,11000余人死亡,引起了国际性的社会恐慌。美、英、法等国家及数个援助机构、国际组织均投入巨额的人力、物力用以抵御西非疫情。我国也先后派出了包括移动式P3实验室检测队、固定实验室检测队、医疗队在内的数支救援力量用于帮助疫区国家和人民,并御疫情于国门之外。埃博拉病毒潜伏期为2-21天,早期症状和一般临床实验检查指标缺乏特异性,而且在非洲地区有许多早期临床症状相似的传染病,如疟疾、伤寒、黄热病等。目前主要有依托ELISA法进行抗原抗体的检测、活病毒培养电子显微镜下观察和RT-PCR核酸检测等方法对埃博拉病毒感染进行确诊。但由于西非受疫情影响最重的几个国家的卫生系统并不健全,缺乏人力和基础设施资源,对于需求昂贵精密仪器设备的现有检测方法难以开展,急需要一种高效、使用方便、不需要精密仪器设备的埃博拉病毒检测方法。LAMP是一种一步法核酸检测技术。自LAMP出现以来,因其反应时间短、操作便捷、不需要精密仪器等特点而成为核酸检测和诊断领域研究的热点,受到了WHO、学界和政府相关部门的广泛关注,短短几年,LAMP被广泛应用于各种病原体的检测。LAMP技术的反应原理是:根据目的基因设计的6条引物靶向性识别目的基因上的六个独立片段,在Bst大片段聚合酶的作用下进行扩增反应,大量扩增目标片段的同时也随之产生了大量沉淀(焦磷酸镁),反应管浊度因此发生变化,因此可以通过实时浊度监测来判断反应结果,还可以加入目视荧光染料进行目视检测。LAMP在扩增过程中能靶向性识别目的基因的六个不同的基因片段,因此具有高特异性,并且整个实验是在恒温条件下进行,仅需要稳定热源就能可以进行反应,不需要大型仪器设备。RT-LAMP技术是检测RNA病毒的LAMP技术,在LAMP反应体系中加入逆转录酶,能同时进行逆转录和核酸扩增,这大大缩短了检测RNA病毒的时间。因其具有上述优点,使之尤为适用于医疗卫生条件较差的西非疫情一线,亦可用于缺少专业技术人才且检测任务繁重的我国口岸检疫机构。2014年西非埃博拉疫情归根结底是扎伊尔型埃博拉病毒感染导致埃博拉病毒病引起的,虽然目前仅在非洲流行,但并不排除其传播到其他大洲的可能性,因此快速检测扎伊尔型埃博拉病毒尤为重要。埃博拉病毒生物安全等级为四级,在常规实验条件下无法开展相关检测工作。因此我们基于扎伊尔型埃博拉病毒NP蛋白的构建了假病毒作为核酸检测的标准品,可代替扎伊尔型埃博拉活毒成为检测靶标。这为扎伊尔型埃博拉病毒的核酸检测方法的建立奠定了良好的基础。扎伊尔型埃博拉的NP基因是核衣壳的编码基因,是一段高度保守的序列。针对扎伊尔型埃博拉的部分NP基因(633bp)设计5套引物,比较5套引物扩增效率得到最佳引物组合,并优化了引物组合的浓度配比。我们采用2种检测方法进行RT-LAMP即实时浊度仪监测法和目视检测法。两种RT-LAMP检测方法均能在61℃恒温条件50min内完成反应(最低检出浓度均为4.56 copies/μl),敏感性是RT-PCR的10倍(RT-PCR为45.6 copies/μl)。此外,我们还同时采用25种非埃博拉病毒和细菌进行特异性检测,结果表明我们建立的RT-LAMP法对扎伊尔型埃博拉病毒具有极佳的特异性。另外,通过临床模拟样本实验可以看出不同的临床样本中的杂质对本方法的敏感性基本没有影响,说明本方法对于样本纯度要求不高且具有良好的稳定性,可应用于基础卫生医疗条件较差的西非各国和我国的基层检测、检疫机构。基于已建立的扎伊尔埃博拉检测方法,我们联合北京爱普益生物科技公司研制了扎伊尔型埃博拉病毒核酸检测(RT-LAMP法)试剂盒。主要用于血液或咽拭子标本RNA中扎伊尔型埃博拉病毒的定性检测,有利于该病毒的辅助诊断及流行病学检测。该试剂盒具有高敏感性,最低检测限达到1×103copies/ml,能耐受"f5天长途运输,反复冻融"f6次性能稳定。并被我国援助塞拉利昂实验室第二批检测队带到塞拉利昂,作为样本初筛的手段。经过一定数量活毒样本检测,并与国际认可的荧光定量RT-PCR试剂盒检测对比,证实了其具有高度的敏感性和特异性。本研究以扎伊尔型埃博拉病毒部分NP基因(633bp)为目的基因,基于RT-LAMP的2种检测方法即实时浊度仪监测法和目视检测法建立了检测扎伊尔型埃博拉病毒方法。基于目视检测法组装了4320人次的试剂盒,其具有可与金标准荧光定量RT-PCR方法基本一致的检测效率,且具有操作简单、无需精密仪器等优点,可用于在疫区一线和缺少专业人才、设备的区域进行扎伊尔型埃博拉病毒染病者的初筛工作,以便在发现阳性病例时便能尽早采取隔离、治疗等措施,以便更好的控制疫情规模和降低疫情造成的损失,最终提高应对埃博拉这一新发、突发烈性传染病的能力。
[Abstract]:Ebola virus (Ebola virus), one of the strong pathogens, has attracted worldwide attention in 2014. The Ebola virus was the first to occur in the two outbreaks of the Ebola region in West Africa in 1976, so it was named Ebola.2014, and Zaire Ebola virus ravaged West Africa, the most since SARS. .WHO, a serious global public health event, determines that the Ebola epidemic in West Africa is the most extensive and complex Ebola outbreak since the outbreak of Ebola in 1976. The number of infections and deaths in the epidemic exceeded all other outbreaks. The Ebola virus spread quickly and first appeared. In the Guinea Republic of West Africa, the post spread to Sierra Leone and Liberia, its border with Nigeria and the United States, and then spread to Mali and Senegal. The epidemic in 9 countries such as West Africa, Europe and the Americas caused more than 29000 people, more than 11000 people died, and international social panic. Beauty, The international organizations, such as Britain, France and other countries, have invested huge manpower and material resources to resist the West African epidemic. China has also sent several rescue forces, including the mobile P3 laboratory testing team, the fixed laboratory testing team, and the medical team to help the countries and people in the epidemic area, and resist the epidemic in the country outside the country. The incubation period of the virus is 2-21 days, the early symptoms and the general clinical test indicators are lack of specificity, and there are many early clinical symptoms similar in Africa, such as malaria, typhoid, yellow fever and so on. At present, it is mainly based on the ELISA method for the detection of antigen antibody, the observation of the live virus culture under the electron microscope and the RT-PCR nucleic acid test. But because of the unsound health systems in the most heavily affected countries of West Africa, lack of human and infrastructure resources, the existing testing methods for expensive and sophisticated equipment are difficult to carry out, and a high efficiency, convenient use and no precision instrument equipment are urgently needed. Ebola virus detection method.LAMP is a one step method of nucleic acid detection. Since the emergence of LAMP, it has become a hot spot in the field of nucleic acid detection and diagnosis because of its short reaction time, convenient operation and no need of precision instruments. It has been widely concerned by WHO, academia and government related departments. In a few years, LAMP has been widely used. The reaction principle of.LAMP technique for detection of various pathogens is that six independent fragments of target genes identified by 6 primers designed by the target gene are amplified by Bst large fragment polymerase, and a large number of target fragments are amplified and a large number of precipitates are produced (magnesium pyrophosphate), and the turbidity of the reaction tube is therefore therefore The results can be judged by real time turbidimetric monitoring, and visual fluorescent dyes can also be added to the visual detection of six different gene fragments that can target the target gene to identify the target gene in the process of amplification. Therefore, the.LAMP has a high specificity, and the whole test is carried out under the constant temperature condition and only needs stable heat source. It can be reacted without the need for large instrument.RT-LAMP technology to detect the RNA virus LAMP technology, and to add reverse transcriptase in the LAMP reaction system to reverse transcription and nucleic acid amplification at the same time, which greatly reduces the time for detecting the RNA virus. The Zaire Ebola epidemic in West Africa, which is not the frontline of the epidemic, is also in the final analysis of the Zaire Ebola virus infection caused by Ebola virus infection of the Ebola virus in the final analysis of the Zaire port quarantine agency, although it is currently in Africa only, but does not exclude the possibility of spreading to other continents. This rapid detection of Zaire Ebola virus is particularly important. The Ebola virus biosafety level is four level and can not be tested under conventional experimental conditions. Therefore, we built a pseudo virus based on the NP protein of Zaire Ebola virus as a marker for nucleic acid detection, instead of Zaire type Ebola. In order to detect the target, it has laid a good foundation for the establishment of the nucleic acid detection method of Zaire Ebola virus. The NP gene of Zaire Ebola is a coding gene of the nucleocapsid, which is a highly conservative sequence. 5 sets of primers are set for the partial NP gene (633bp) of Ebola of Zaire type, and the amplification efficiency of 5 sets of primers is compared. The best primer combination was used and the concentration ratio of primer combinations was optimized. We used 2 detection methods to perform RT-LAMP real-time turbidimeter monitoring and visual detection. The two RT-LAMP detection methods were able to complete the reaction at a constant temperature of 61 degrees centigrade 50min (the lowest detection concentration was 4.56 copies/ Mu L), and the sensitivity was 10 times of RT-PCR (RT-PCR was 45.6). In addition, we also used 25 kinds of non Ebola virus and bacteria to carry out specific tests. The results show that the RT-LAMP method established by us has excellent specificity for Zaire Ebola virus. In addition, we can see the sensitivity of the impurities in the different bed samples to this method by the clinical simulation sample experiment. This method has no effect on the purity of the sample and has good stability. It can be applied to the West African countries with poor basic health care conditions and the grass-roots inspection and quarantine institutions in our country. Based on the established Zaire Ebola testing method, we have developed the Zaire type angstrom with the Beijing EPI biotechnology company. Bora virus nucleic acid detection (RT-LAMP) kit. It is mainly used in the qualitative detection of Zaire type Ebola virus in blood or swab specimens, which is beneficial to the auxiliary diagnosis and epidemiological detection of the virus. The kit has Gao Min sensibility, the minimum detection limit reaches 1 x 103copies/ml, and can tolerate "F5 days long-distance transportation, repeated freezing and thawing" F6 The secondary performance was stable and was brought to Sierra Leone by the second group of Sierra Leone laboratories in China. It was used as a sample screening method. A certain number of live virus samples were tested and compared with the internationally recognized fluorescent quantitative RT-PCR kits, which proved to be highly sensitive and specific. This study was based on Zaire EBO. The partial NP gene (633bp) of the virus is the target gene, and the detection method of Zaire type Ebola virus is established based on 2 detection methods of RT-LAMP, namely, real time turbidimeter monitoring and visual inspection. 4320 times of the kit are assembled based on visual inspection. It has the same detection effect as the gold standard quasi fluorescence quantitative RT-PCR method. It has the advantages of simple operation and no precision instrument. It can be used in the first screening work of the Zaire Ebola virus infected people in the front-line of the epidemic area and the lack of professional personnel and equipment, so that isolation and treatment can be taken as early as possible in the discovery of positive cases, so as to better control the scale of the epidemic and reduce the epidemic situation. The loss will ultimately enhance the ability to deal with the new outbreak of Ebola and sudden infectious diseases.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.8

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