鸡传染性贫血病毒感染宿主后的基因表达谱分析及实时荧光定量PCR检测方法的建立
发布时间：2018-07-31 14:38
【摘要】：鸡传染性贫血病毒(chicken infectious anemia virus,CIAV)能够引起再生障碍性贫血以及胸腺和法氏囊等淋巴组织的萎缩,已成为造成养鸡业巨大经济损失的重要原因之一。基因表达谱芯片属于基因芯片的一种,近年来得到了广泛应用,它能够用来检测宿主的基因组,研究病毒在不同状态下的基因表达情况。实时荧光定量PCR的原理是通过PCR产物的累积,使荧光信号强度等比例增加来实时监控扩增产物。本文研究了两部分,第一部分为鸡感染鸡传染性贫血病毒后的表达谱分析,第二部分为实时荧光定量PCR检测方法的建立。1.鸡感染鸡传染性贫血病毒后的表达谱分析由于鸡传染性贫血病毒的主要感染对象是雏鸡,并且能够通过水平传播和垂直传播,从而造成鸡只大量死亡。为了深入了解其致病机制,本实验首先通过CIAV感染1日龄SPF雏鸡进行动物模型的建立,同时设立阴性对照,结果发现鸡只感染病毒后14日时发病最严重、症状最明显,于是采取14日龄鸡只胸腺,提取总RNA,反转录成c DNA,在体外合成c RNA,采用Affymetrix全基因组表达谱芯片与其杂交,从而得到基因表达谱的变化情况。利用SAM软件进行差异表达基因的筛选,同时将这些差异表达基因进行聚类分析,之后通过DAVID软件对我们得到的差异表达基因进行生物学功能分析,同时应用实时荧光定量PCR对基因表达谱芯片得到的结果进行验证。结果成功筛选出13708个差异表达基因,其中1759个上调基因,529个下调基因,其中与免疫相关的基因有38个,11420个基因未发生变化。GO和Pathway分析结果显示,这些差异表达基因主要与免疫应答、细胞周期、细胞凋亡、血小板凝集等重要的信号通路有关。我们采用实时荧光定量PCR技术随机验证其中的7个差异表达基因,得到的结果与基因表达谱芯片的结果具有一致性,表明这次的芯片实验结果具有可靠性。2、实时荧光定量PCR检测方法的建立实时荧光定量PCR对样品检测准确、灵敏,并且能够准确定量,弥补了病毒分离鉴定、血清学诊断、PCR诊断方法、电镜检查以及测量血细胞压积值等检测方法的不足。实时荧光定量PCR包括探针法和染料法,由于SYBR GreenⅠ染料操作简单,成本低廉,因此得到广泛应用。本试验根据CIAV基因组的保守区域设计了1对特异性引物,扩增后得到的片段大小为180bp,制备PGM-T-CIAV重组质粒,得到阳性质粒的标准品,绘制Q-RT PCR标准方程曲线,并做敏感性试验、特异性试验和重复性试验,最后对临床样品进行了检测。试验结果证实,在进行CIAV检测时,Q-RT PCR比普通PCR灵敏度要高、特异性要强、重复性要好,并且在对临床样品进行检验时,再一次证明其具有很高的灵敏度。
[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，
本文编号：2155901
[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，
本文编号：2155901
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