發(fā)布時間：2018-06-21 14:59

  本文選題：扎伊爾型埃博拉病毒 + 環(huán)介導(dǎo)恒溫擴(kuò)增技術(shù)�。� 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文

【摘要】：埃博拉病毒(Ebola virus)作為烈性病原體之一,在2014年的暴發(fā)流行引起了全球的關(guān)注。埃博拉病毒是最早出現(xiàn)在1976年暴發(fā)于西非埃博拉河地區(qū)兩起疫情中,因此被命名為埃博拉。2014年,扎伊爾型埃博拉病毒病肆虐西非,其為自SARS以來最為嚴(yán)重的全球性公共衛(wèi)生事件。WHO判定此次西非埃博拉疫情是自1976年埃博拉病毒被發(fā)現(xiàn)以來暴發(fā)的范圍最廣且最復(fù)雜的埃博拉疫情,這次疫情出現(xiàn)的感染人數(shù)和死亡人數(shù)超出了其它全部疫情的總和。埃博拉病毒傳播速度極快,首先出現(xiàn)在西非的幾內(nèi)亞共和國,后傳播至與其接壤的塞拉利昂和利比里亞,又由空中輸入尼日利亞和美國,然后蔓延到馬里和塞內(nèi)加爾。此次疫情在西非、歐洲和美洲等9個國家共造成了29000余人感染,11000余人死亡,引起了國際性的社會恐慌。美、英、法等國家及數(shù)個援助機(jī)構(gòu)、國際組織均投入巨額的人力、物力用以抵御西非疫情。我國也先后派出了包括移動式P3實驗室檢測隊、固定實驗室檢測隊、醫(yī)療隊在內(nèi)的數(shù)支救援力量用于幫助疫區(qū)國家和人民,并御疫情于國門之外。埃博拉病毒潛伏期為2-21天,早期癥狀和一般臨床實驗檢查指標(biāo)缺乏特異性,而且在非洲地區(qū)有許多早期臨床癥狀相似的傳染病,如瘧疾、傷寒、黃熱病等。目前主要有依托ELISA法進(jìn)行抗原抗體的檢測、活病毒培養(yǎng)電子顯微鏡下觀察和RT-PCR核酸檢測等方法對埃博拉病毒感染進(jìn)行確診。但由于西非受疫情影響最重的幾個國家的衛(wèi)生系統(tǒng)并不健全,缺乏人力和基礎(chǔ)設(shè)施資源,對于需求昂貴精密儀器設(shè)備的現(xiàn)有檢測方法難以開展,急需要一種高效、使用方便、不需要精密儀器設(shè)備的埃博拉病毒檢測方法。LAMP是一種一步法核酸檢測技術(shù)。自LAMP出現(xiàn)以來,因其反應(yīng)時間短、操作便捷、不需要精密儀器等特點而成為核酸檢測和診斷領(lǐng)域研究的熱點,受到了WHO、學(xué)界和政府相關(guān)部門的廣泛關(guān)注,短短幾年,LAMP被廣泛應(yīng)用于各種病原體的檢測。LAMP技術(shù)的反應(yīng)原理是:根據(jù)目的基因設(shè)計的6條引物靶向性識別目的基因上的六個獨(dú)立片段,在Bst大片段聚合酶的作用下進(jìn)行擴(kuò)增反應(yīng),大量擴(kuò)增目標(biāo)片段的同時也隨之產(chǎn)生了大量沉淀(焦磷酸鎂),反應(yīng)管濁度因此發(fā)生變化,因此可以通過實時濁度監(jiān)測來判斷反應(yīng)結(jié)果,還可以加入目視熒光染料進(jìn)行目視檢測。LAMP在擴(kuò)增過程中能靶向性識別目的基因的六個不同的基因片段,因此具有高特異性,并且整個實驗是在恒溫條件下進(jìn)行,僅需要穩(wěn)定熱源就能可以進(jìn)行反應(yīng),不需要大型儀器設(shè)備。RT-LAMP技術(shù)是檢測RNA病毒的LAMP技術(shù),在LAMP反應(yīng)體系中加入逆轉(zhuǎn)錄酶,能同時進(jìn)行逆轉(zhuǎn)錄和核酸擴(kuò)增,這大大縮短了檢測RNA病毒的時間。因其具有上述優(yōu)點,使之尤為適用于醫(yī)療衛(wèi)生條件較差的西非疫情一線,亦可用于缺少專業(yè)技術(shù)人才且檢測任務(wù)繁重的我國口岸檢疫機(jī)構(gòu)。2014年西非埃博拉疫情歸根結(jié)底是扎伊爾型埃博拉病毒感染導(dǎo)致埃博拉病毒病引起的,雖然目前僅在非洲流行,但并不排除其傳播到其他大洲的可能性,因此快速檢測扎伊爾型埃博拉病毒尤為重要。埃博拉病毒生物安全等級為四級,在常規(guī)實驗條件下無法開展相關(guān)檢測工作。因此我們基于扎伊爾型埃博拉病毒NP蛋白的構(gòu)建了假病毒作為核酸檢測的標(biāo)準(zhǔn)品,可代替扎伊爾型埃博拉活毒成為檢測靶標(biāo)。這為扎伊爾型埃博拉病毒的核酸檢測方法的建立奠定了良好的基礎(chǔ)。扎伊爾型埃博拉的NP基因是核衣殼的編碼基因,是一段高度保守的序列。針對扎伊爾型埃博拉的部分NP基因(633bp)設(shè)計5套引物,比較5套引物擴(kuò)增效率得到最佳引物組合,并優(yōu)化了引物組合的濃度配比。我們采用2種檢測方法進(jìn)行RT-LAMP即實時濁度儀監(jiān)測法和目視檢測法。兩種RT-LAMP檢測方法均能在61℃恒溫條件50min內(nèi)完成反應(yīng)(最低檢出濃度均為4.56 copies/μl),敏感性是RT-PCR的10倍(RT-PCR為45.6 copies/μl)。此外,我們還同時采用25種非埃博拉病毒和細(xì)菌進(jìn)行特異性檢測,結(jié)果表明我們建立的RT-LAMP法對扎伊爾型埃博拉病毒具有極佳的特異性。另外,通過臨床模擬樣本實驗可以看出不同的臨床樣本中的雜質(zhì)對本方法的敏感性基本沒有影響,說明本方法對于樣本純度要求不高且具有良好的穩(wěn)定性,可應(yīng)用于基礎(chǔ)衛(wèi)生醫(yī)療條件較差的西非各國和我國的基層檢測、檢疫機(jī)構(gòu)。基于已建立的扎伊爾埃博拉檢測方法,我們聯(lián)合北京愛普益生物科技公司研制了扎伊爾型埃博拉病毒核酸檢測(RT-LAMP法)試劑盒。主要用于血液或咽拭子標(biāo)本RNA中扎伊爾型埃博拉病毒的定性檢測,有利于該病毒的輔助診斷及流行病學(xué)檢測。該試劑盒具有高敏感性,最低檢測限達(dá)到1×103copies/ml,能耐受"f5天長途運(yùn)輸,反復(fù)凍融"f6次性能穩(wěn)定。并被我國援助塞拉利昂實驗室第二批檢測隊帶到塞拉利昂,作為樣本初篩的手段。經(jīng)過一定數(shù)量活毒樣本檢測,并與國際認(rèn)可的熒光定量RT-PCR試劑盒檢測對比,證實了其具有高度的敏感性和特異性。本研究以扎伊爾型埃博拉病毒部分NP基因(633bp)為目的基因,基于RT-LAMP的2種檢測方法即實時濁度儀監(jiān)測法和目視檢測法建立了檢測扎伊爾型埃博拉病毒方法�；谀恳暀z測法組裝了4320人次的試劑盒,其具有可與金標(biāo)準(zhǔn)熒光定量RT-PCR方法基本一致的檢測效率,且具有操作簡單、無需精密儀器等優(yōu)點,可用于在疫區(qū)一線和缺少專業(yè)人才、設(shè)備的區(qū)域進(jìn)行扎伊爾型埃博拉病毒染病者的初篩工作,以便在發(fā)現(xiàn)陽性病例時便能盡早采取隔離、治療等措施,以便更好的控制疫情規(guī)模和降低疫情造成的損失,最終提高應(yīng)對埃博拉這一新發(fā)、突發(fā)烈性傳染病的能力。
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.8

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 張健;蘇海濱;;埃博拉病毒病臨床特征與治療[J];傳染病信息;2016年01期

2 朱祥;堯晨光;魏艷紅;寇錚;胡康洪;;埃博拉疫苗和藥物研究進(jìn)展[J];病毒學(xué)報;2015年03期

3 曾谷城;;埃博拉病毒研究進(jìn)展[J];中山大學(xué)學(xué)報(醫(yī)學(xué)科學(xué)版);2015年02期

4 程穎;劉軍;李昱;劉翟;任翔;施一;高福;余宏杰;;埃博拉病毒病:病原學(xué)、致病機(jī)制、治療與疫苗研究進(jìn)展[J];科學(xué)通報;2014年30期

5 張云輝;王姝;陳玉琪;李軍;;埃博拉出血熱研究現(xiàn)狀及2014年疫情進(jìn)展[J];傳染病信息;2014年04期

6 李昱;任翔;劉翟;程穎;高福;余宏杰;;埃博拉病毒病:流行病學(xué)、生態(tài)學(xué)、診斷、治療及控制[J];科技導(dǎo)報;2014年24期

7 Windell L.Rivera;Vanissa A.Ong;;Development of loop-mediated isothermal amplification for rapid detection of Entamoeba histolytica[J];Asian Pacific Journal of Tropical Medicine;2013年06期

8 王曉杜;劉陽;王皓婷;史子學(xué);趙凡凡;魏建超;邵東華;馬志永;;埃博拉病毒NP蛋白的單克隆抗體制備及抗原肽的定位[J];生物工程學(xué)報;2012年11期

9 劉威;鄒大陽;尹志濤;劉大偉;李雪蓮;魏曉;王雪松;姜錚;袁靜;黃留玉;;基于顏色判定的環(huán)介導(dǎo)恒溫擴(kuò)增法快速檢測副溶血性弧菌[J];中國科學(xué):生命科學(xué);2011年10期

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