本文選題：小反芻獸疫 + 免疫過(guò)氧化物酶單層試驗(yàn)��； 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：小反芻獸疫(Peste des petits ruminants,PPR)是由副粘病毒科麻疹病毒屬的小反芻獸疫病毒(Pest des petits ruminants virus,PPRV)引起的一種急性、高度接觸性烈性傳染病,主要感染山羊和綿羊等小反芻動(dòng)物。PPR自1942年在非洲被發(fā)現(xiàn)以來(lái),不斷向全球蔓延。2007年7月,我國(guó)首次在西藏爆發(fā)PPR疫情,2013年至2014年全國(guó)19個(gè)省區(qū)再次爆發(fā)PPR疫情,對(duì)我國(guó)養(yǎng)殖業(yè)造成巨大經(jīng)濟(jì)損失。2015年4月,在科特迪瓦首都阿比讓舉行的動(dòng)物衛(wèi)生專(zhuān)家會(huì)議一致同意,嘗試在全球消除小反芻獸疫病毒。因此,盡快開(kāi)展小反芻獸疫的病原學(xué)、流行病學(xué)、診斷與預(yù)防控制技術(shù)的綜合性研究,建立快速敏感的診斷方法對(duì)PPR的防控具有深遠(yuǎn)的社會(huì)和經(jīng)濟(jì)意義。目前針對(duì)PPRV的診斷方法較多,其中血清學(xué)檢測(cè)方法主要是病毒中和試驗(yàn)(virus neutralization test,VNT)和競(jìng)爭(zhēng)ELISA方法,但前者存在操作周期長(zhǎng),因操作活病毒而設(shè)施要求高等缺點(diǎn);后者存在價(jià)格較貴、制備復(fù)雜等缺點(diǎn)。免疫過(guò)氧化酶單層試驗(yàn)(immunoperoxidase monolayer assay,IPMA)具有快速、敏感、特異、操作簡(jiǎn)單的特點(diǎn),已應(yīng)用于多種病原的血清臨床樣本檢測(cè)。但國(guó)內(nèi)外還沒(méi)有建立針對(duì)PPRV的IPMA檢測(cè)方法,因此建立PPRV-IPMA檢測(cè)方法對(duì)于PPR疫情的監(jiān)測(cè)和防控具有一定的重要意義。為建立針對(duì)PPR的IPMA檢測(cè)方法,本研究首先建立了穩(wěn)定表達(dá)山羊SLAM的BHK-21細(xì)胞系(BHK-gSLAM)。rPPRV/GFP感染該細(xì)胞系后,能復(fù)制增殖且形成明顯的合胞體,而在親本細(xì)胞上無(wú)該現(xiàn)象。Western blot試驗(yàn)表明該細(xì)胞系能穩(wěn)定表達(dá)SLAM蛋白至少20代。在細(xì)胞系建立的基礎(chǔ)上,通過(guò)感染rPPRV/GFP制備抗原檢測(cè)板,建立了IPMA檢測(cè)方法,具體步驟如下:首先比較了Vero細(xì)胞和BHK-gSLAM兩者在IPMA試驗(yàn)中的差別,結(jié)果表明,在IPMA反應(yīng)板制備過(guò)程中,同等劑量的rPPRV/GFP感染Vero和BHK-gSLAM細(xì)胞,后者病變形成時(shí)間更早,并形成易于觀(guān)察的合胞體;BHK-gSLAM細(xì)胞生長(zhǎng)更緊密,形成的CPE更明顯,其在AEC染色后更易于觀(guān)察,因此最終選擇該細(xì)胞系用于IPMA抗原檢測(cè)板的制備。其次,本研究?jī)?yōu)化并確定了IPMA的最佳接毒劑量為104 TCID50/mL,病毒感染72 h后固定制備IPMA反應(yīng)板,血清最佳起始稀釋度為1:10,二抗最佳工作濃度為1:5000。最后,用該IPMA檢測(cè)方法對(duì)198份羊血清(包括臨床樣本和PPR弱毒疫苗免疫后樣本)進(jìn)行檢測(cè),同時(shí)以VNT方法作為對(duì)照,結(jié)果表明,兩者的符合率為95.5%。與VNT相比,IPMA的相對(duì)敏感性為91%,特異性為100%。綜上所述,本研究建立的IPMA方法操作簡(jiǎn)單,易于觀(guān)察,檢測(cè)時(shí)間僅為3小時(shí),檢測(cè)結(jié)果與VNT方法符合率較高,同時(shí)具有良好的特異性和敏感性,可以替代VNT用于PPRV免疫效果的評(píng)價(jià)和流行病學(xué)調(diào)查。
[Abstract]:Peste des petits ruminants (PPR) is an acute and highly contagious disease caused by the measles virus of the genus Pest des petits ruminants virus (PPRV). PPR, which mainly infects small ruminants such as goats and sheep, has been spreading to the world since it was discovered in Africa in 1942. In July 2007, the PPR epidemic occurred for the first time in China in Tibet, and again in 19 provinces and regions in China from 2013 to 2014. In April 2015, an expert meeting on animal health in Abidjan, Ivory Coast, agreed to try to eliminate the small ruminant virus globally. Therefore, it is of great social and economic significance to develop a comprehensive study on the etiology, epidemiology, diagnosis and prevention and control of small ruminant disease as soon as possible, and to establish a rapid and sensitive diagnostic method for the prevention and control of PPR. At present, there are many diagnostic methods for PPRV, of which the serological detection methods are mainly virus neutralization test (virus neutralization testVNT) and competitive Elisa. However, the former has the disadvantages of long operation cycle and high facility requirements due to the operation of live virus. Complex preparation and other shortcomings. The immunoperoxidase monolayer test (immunoperoxidase monolayer assayma) has been applied to the detection of various pathogens in serum because of its rapidity, sensitivity, specificity and simple operation. However, there is no IPMA detection method for PPRV at home and abroad, so it is of great significance to establish PPRV-IPMA detection method for PPR epidemic situation monitoring and prevention and control. In order to establish IPMA detection method for PPR, BHK-21 cell line (BHK-gSLAM), rPPRV / GFP, which expressed goat slam stably, could replicate and proliferate and form distinct syncytial cells after infecting the cell line. Western blot assay showed that the cell line could stably express slam protein for at least 20 passages. Based on the establishment of the cell line, the antigen detection plate was prepared by rPPRV / GFP infection, and the IPMA detection method was established. The specific steps are as follows: firstly, the differences between Vero cells and BHK-gSLAM in IPMA test were compared. The results showed that, during the preparation of IPMA reaction plate, the difference between Vero cells and BHK-gSLAM in IPMA test was compared. The same dose of rPPRV / GFP was infected with Vero and BHK-gSLAM cells, the latter developed earlier, and formed the easily observable syncytial cells BHK-gSLAM cells. The formation of CPE was more obvious, and it was easier to observe after AEC staining. Therefore, the cell line was selected for the preparation of IPMA antigen detection plate. Secondly, the optimal dose of IPMA was 104TCID 50 / mL. The IPMA reaction plate was fixed at 72 h after infection. The optimal initial dilution of IPMA was 1: 10, and the optimal working concentration of the second antibody was 1: 5000. Finally, 198 sheep serum samples (including clinical samples and PPR attenuated vaccine samples) were detected by the IPMA method, and the VNT method was used as control. The results showed that the coincidence rate of the two methods was 95.55g. Compared with VNT, the relative sensitivity of IPMA was 91 and the specificity was 100. In conclusion, the IPMA method established in this study is simple to operate, easy to observe, and the detection time is only 3 hours. The detection results are in good agreement with the VNT method and have good specificity and sensitivity. VNT can be used to evaluate the immune effect of PPRV and to investigate the epidemiology of PPRV.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.4

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相關(guān)期刊論文 前1條

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本文編號(hào)：2061585