【摘要】：雞傳染性貧血病毒(chicken infectious anemia virus,CIAV)能夠引起再生障礙性貧血以及胸腺和法氏囊等淋巴組織的萎縮,已成為造成養(yǎng)雞業(yè)巨大經(jīng)濟(jì)損失的重要原因之一�；虮磉_(dá)譜芯片屬于基因芯片的一種,近年來(lái)得到了廣泛應(yīng)用,它能夠用來(lái)檢測(cè)宿主的基因組,研究病毒在不同狀態(tài)下的基因表達(dá)情況。實(shí)時(shí)熒光定量PCR的原理是通過(guò)PCR產(chǎn)物的累積,使熒光信號(hào)強(qiáng)度等比例增加來(lái)實(shí)時(shí)監(jiān)控?cái)U(kuò)增產(chǎn)物。本文研究了兩部分,第一部分為雞感染雞傳染性貧血病毒后的表達(dá)譜分析,第二部分為實(shí)時(shí)熒光定量PCR檢測(cè)方法的建立。1.雞感染雞傳染性貧血病毒后的表達(dá)譜分析由于雞傳染性貧血病毒的主要感染對(duì)象是雛雞,并且能夠通過(guò)水平傳播和垂直傳播,從而造成雞只大量死亡。為了深入了解其致病機(jī)制,本實(shí)驗(yàn)首先通過(guò)CIAV感染1日齡SPF雛雞進(jìn)行動(dòng)物模型的建立,同時(shí)設(shè)立陰性對(duì)照,結(jié)果發(fā)現(xiàn)雞只感染病毒后14日時(shí)發(fā)病最嚴(yán)重、癥狀最明顯,于是采取14日齡雞只胸腺,提取總RNA,反轉(zhuǎn)錄成c DNA,在體外合成c RNA,采用Affymetrix全基因組表達(dá)譜芯片與其雜交,從而得到基因表達(dá)譜的變化情況。利用SAM軟件進(jìn)行差異表達(dá)基因的篩選,同時(shí)將這些差異表達(dá)基因進(jìn)行聚類分析,之后通過(guò)DAVID軟件對(duì)我們得到的差異表達(dá)基因進(jìn)行生物學(xué)功能分析,同時(shí)應(yīng)用實(shí)時(shí)熒光定量PCR對(duì)基因表達(dá)譜芯片得到的結(jié)果進(jìn)行驗(yàn)證。結(jié)果成功篩選出13708個(gè)差異表達(dá)基因,其中1759個(gè)上調(diào)基因,529個(gè)下調(diào)基因,其中與免疫相關(guān)的基因有38個(gè),11420個(gè)基因未發(fā)生變化。GO和Pathway分析結(jié)果顯示,這些差異表達(dá)基因主要與免疫應(yīng)答、細(xì)胞周期、細(xì)胞凋亡、血小板凝集等重要的信號(hào)通路有關(guān)。我們采用實(shí)時(shí)熒光定量PCR技術(shù)隨機(jī)驗(yàn)證其中的7個(gè)差異表達(dá)基因,得到的結(jié)果與基因表達(dá)譜芯片的結(jié)果具有一致性,表明這次的芯片實(shí)驗(yàn)結(jié)果具有可靠性。2、實(shí)時(shí)熒光定量PCR檢測(cè)方法的建立實(shí)時(shí)熒光定量PCR對(duì)樣品檢測(cè)準(zhǔn)確、靈敏,并且能夠準(zhǔn)確定量,彌補(bǔ)了病毒分離鑒定、血清學(xué)診斷、PCR診斷方法、電鏡檢查以及測(cè)量血細(xì)胞壓積值等檢測(cè)方法的不足。實(shí)時(shí)熒光定量PCR包括探針?lè)ê腿玖戏?由于SYBR GreenⅠ染料操作簡(jiǎn)單,成本低廉,因此得到廣泛應(yīng)用。本試驗(yàn)根據(jù)CIAV基因組的保守區(qū)域設(shè)計(jì)了1對(duì)特異性引物,擴(kuò)增后得到的片段大小為180bp,制備PGM-T-CIAV重組質(zhì)粒,得到陽(yáng)性質(zhì)粒的標(biāo)準(zhǔn)品,繪制Q-RT PCR標(biāo)準(zhǔn)方程曲線,并做敏感性試驗(yàn)、特異性試驗(yàn)和重復(fù)性試驗(yàn),最后對(duì)臨床樣品進(jìn)行了檢測(cè)。試驗(yàn)結(jié)果證實(shí),在進(jìn)行CIAV檢測(cè)時(shí),Q-RT PCR比普通PCR靈敏度要高、特異性要強(qiáng)、重復(fù)性要好,并且在對(duì)臨床樣品進(jìn)行檢驗(yàn)時(shí),再一次證明其具有很高的靈敏度。
[Abstract]:Chicken infectious anemia virus (chicken infectious anemia virus) can cause aplastic anemia and the atrophy of thymus and bursa of Fabricius, which has become one of the important reasons for the great economic loss of chicken industry. Gene expression microarray is a kind of gene chip, which has been widely used in recent years. It can be used to detect the host genome and study the gene expression of virus in different states. The principle of real-time fluorescence quantitative PCR is to monitor the amplification product by the accumulation of PCR products and the increase of fluorescence signal intensity. Two parts were studied in this paper. The first part was the analysis of the expression profile of chicken infectious anemia virus. The second part was the establishment of real-time fluorescent quantitative PCR detection method. Expression profile Analysis of Chicken Infectious anemia virus the main infection object of chicken infectious anemia virus is chicks and can be spread horizontally and vertically resulting in a large number of chicken deaths. In order to understand the pathogenetic mechanism of SPF chicks infected with CIAV at first, the animal model was established by CIAV infection for 1 day, and negative control was set up. The results showed that the disease was the most serious and the symptoms were the most obvious at 14 days after infection. The total RNAs were extracted from the thymus of 14-day-old chickens, and then reverse transcribed into c DNAs. The cDNA was synthesized in vitro and hybridized with the whole genome expression microarray of Affymetrix, and the variation of gene expression profile was obtained. SAM software was used to screen differentially expressed genes, and these differentially expressed genes were analyzed by cluster analysis, and then the biological functions of the differentially expressed genes were analyzed by DAVID software. At the same time, real-time fluorescence quantitative PCR was used to verify the results obtained by gene expression microarray. Results 13708 differentially expressed genes were successfully screened, of which 1759 up-regulated genes were identified as 529 down-regulated genes, 38 of which were immune-related genes. The results of .go and Pathway analysis showed that there was no change in 11420 genes. These differentially expressed genes are mainly related to immune response, cell cycle, apoptosis, platelet agglutination and other important signal pathways. Seven differentially expressed genes were randomly verified by real-time fluorescent quantitative PCR, and the results were consistent with those obtained by gene expression microarray. The results show that the chip experiment results are reliable. The method of real-time fluorescence quantitative PCR detection is accurate, sensitive and accurate, which makes up for the isolation and identification of virus. The methods of serological diagnosis and PCR, electron microscopy and hematocrit measurement were insufficient. Real-time fluorescent quantitative PCR includes probe method and dye method. Because of its simple operation and low cost, SYBR Green 鈪，

本文編號(hào)：2155901