發(fā)布時間：2018-05-10 06:19

  本文選題：東部、西部馬腦脊髓炎病毒 + 抗體��； 參考：《中國農(nóng)業(yè)大學》2015年博士論文

【摘要】：東部、西部馬腦脊髓炎病毒可引起人和動物的嚴重感染,是我國出入境檢驗檢疫中的一類檢疫對象,我國目前尚未有這兩種病的病例報道。隨著馬匹國際貿(mào)易和國際賽事的日益繁榮,來自不同的國家和地區(qū)、健康狀況不同的馬匹可能進口到我國,因此傳入風險不斷加大。對此,除實施嚴格的隔離檢疫制度之外,還需研制快速、準確的檢測技術(shù)作為儲備。本研究針對口岸檢疫實際需求,研究建立了東部、西部馬腦脊髓炎病毒熒光RT-PCR檢測方法和IgM抗體間接ELISA檢測方法并應(yīng)用于口岸臨床樣品的檢測。采用TaqMan方法,在對東部、西部馬腦脊髓炎病毒基因組序列多重比對基礎(chǔ)上,設(shè)計合成簡并引物和探針。通過優(yōu)化各項反應(yīng)條件,建立了兩種分別針對東部、西部馬腦脊髓炎病毒的實時熒光RT-PCR檢測方法。為評價方法的靈敏度,通過體外轉(zhuǎn)錄制備了東部、西部馬腦脊髓炎病毒保守區(qū)序列的核酸標準品cRNA,經(jīng)稀釋、分裝、定量、均勻性和穩(wěn)定性檢驗后,用于方法的靈敏度評價。結(jié)果顯示建立的兩種方法靈敏度均達到10拷貝/反應(yīng)。特異性試驗結(jié)果顯示建立的方法特異,與收集的相關(guān)馬病病原核酸無交叉反應(yīng)。對臨床樣品的檢測中進一步證實研制的熒光RT-PCR檢測方法快速、敏感、特異且重復(fù)性好,檢測時限3個小時以內(nèi)。經(jīng)基因擴增分別獲取了東部、西部馬腦脊髓炎病毒E2蛋白編碼基因,進而將獲得的E2基因分別克隆到原核表達載體,然后轉(zhuǎn)化E.coli Rosseta細胞后進行誘導(dǎo),SDS-PAGE結(jié)果顯示外源基因獲得表達。將表達產(chǎn)物作Western-blot鑒定,結(jié)果表明表達產(chǎn)物能夠分別與東部、西部馬腦脊髓炎病毒IgM陽性血清產(chǎn)生反應(yīng),表明其具有反應(yīng)原性。以純化后的重組蛋白分別包被酶標板,使用東部、西部馬腦脊髓炎病毒IgM陰、陽性血清建立了檢測東部馬腦脊髓炎、西部馬腦脊髓炎IgM抗體的間接ELISA方法。對建立的方法的敏感性、特異性進行評價后,經(jīng)板內(nèi)重復(fù)試驗和板間重復(fù)試驗證明檢測方法重復(fù)性良好,并使用研制的兩種ELISA檢測方法對已知陰、陽性馬血清樣品進行了驗證檢測,檢測結(jié)果與預(yù)期完全一致。
[Abstract]:Equine encephalomyelitis virus (EMDV) in the east and west of China can cause serious infection in human and animal. It is a kind of quarantine object in the entry and exit inspection and quarantine of our country. At present, there are no cases of these two diseases reported in our country. With the increasing prosperity of international trade and race of horses, horses with different health status may be imported into China from different countries and regions, so the risk of import is increasing. Therefore, in addition to strict quarantine and quarantine system, rapid and accurate detection technology should be developed as reserve. In order to meet the actual requirement of quarantine at the port, this study established the method of detecting equine encephalomyelitis virus fluorescence RT-PCR in the east and west of China and the indirect ELISA detection method of IgM antibody, and applied it to the detection of clinical samples at the port. TaqMan method was used to design and synthesize degenerate primers and probes on the basis of multiple alignment of equine encephalomyelitis virus genome sequences in the east and west of China. By optimizing the reaction conditions, two real-time fluorescence RT-PCR detection methods for equine encephalomyelitis virus in eastern and western regions were established. In order to evaluate the sensitivity of the method, the standard nucleic acid (cRNAs) of the conserved region of equine encephalomyelitis virus in the east and west of China were prepared by in vitro transcription. After dilution, loading, quantification, homogeneity and stability tests, cRNAs were used to evaluate the sensitivity of the method. The results showed that the sensitivity of the two methods was 10 copies / reaction. The results of specificity test showed that the method was specific and had no cross-reaction with the collected nucleic acid of horse disease. It was further confirmed that the developed fluorescent RT-PCR method was rapid, sensitive, specific and reproducible, and the detection time was less than 3 hours. The E2 protein encoding gene of equine encephalomyelitis virus (EMDV) was obtained by gene amplification, and then cloned into prokaryotic expression vector. Then the E.coli Rosseta cells were transformed and induced by SDS-PAGE. The expression product was identified by Western-blot. The results showed that the expressed product could react with the IgM positive serum of equine encephalomyelitis virus in the east and west of China respectively, which indicated that the product was reactive. The purified recombinant protein was coated with enzyme-labeled plates, and an indirect ELISA method was established for the detection of IgM antibodies against equine encephalomyelitis and equine encephalomyelitis in eastern and western equine encephalomyelitis by using the positive serum of equine encephalomyelitis virus (IgM) in the east and west of the region. The sensitivity and specificity of the established method were evaluated. The results of intraplate repeated test and inter-plate repeated test proved that the method was reproducible, and two ELISA detection methods were used to detect the known negative. Positive horse serum samples were verified and tested, and the results were in accordance with expectations.
【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S858.21
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本文編號：1868168