新型鸭呼肠孤病毒检测方法及细胞适应性的研究
发布时间：2018-10-30 06:19
【摘要】：新型鸭呼肠孤病毒病是近年来新出现的疫病,主要特征为肝、脾脏不规则出血、坏死与心肌、腔上囊出血,最初被称为“鸭白点病”、“鸭新肝病”、“鸭坏死性肝炎病”等。现已确定该病是由新型鸭呼肠孤病毒引起。为了提高新型鸭呼肠孤病毒感染诊断的准确性、灵敏度和实效性,本研究建立了NDRV荧光定量RT-PCR检测方法和RT-LAMP检测方法。另外,本研究还对该病毒在不同细胞上的适应性进行分析,并将该病毒在鸡胚成纤维细胞(CEF)上进行了克隆纯化与传代,为该病弱毒疫苗的研发奠定了基础。本研究分为三部分:1.NDRV荧光定量RT-PCR检测方法的建立根据NDRV S1基因序列设计了1对特异性引物,建立基于SYBR GreenⅠ的实时荧光定量RT-PCR检测方法。根据含目的基因的质粒拷贝数与定量反应Ct值的关系,绘制了标准曲线。该方法敏感性高,比普通常规PCR高100倍,并具有良好的特异性和重复性。利用该方法对人工感染死亡雏鸭的不同组织器官进行检测,结果发现脾脏和肝脏中的含毒量较高。2.NDRV RT-LAMP检测方法的建立基于NDRV S3基因的6个保守区域设计了4条LAMP引物,利用Bst DNA聚合酶在63℃恒温保持45min即可完成反转录和扩增反应,由此建立了RT-LAMP检测方法。该方法具有良好的特异性,敏感性为常规RT-PCR方法的100倍。临床应用结果表明,该方法与病毒分离鉴定方法的符合率为98%,而且对仪器要求低,适于基层实验室和现场检测。3.NDRV细胞适应性分析将NDRV分别在DEF、CEF、Vero、BHK-21、DF-1等细胞传代至适应稳定后,分别收集不同时间段的病毒液并测定其TCID50,绘制病毒的生长曲线,筛选出鸭新型呼肠孤病毒最适应的细胞为CEF细胞。将NDRV在CEF进行克隆纯化,并进行了传代,病毒滴度由103.7/0.1mL提高到了106.4/0.1mL。
[Abstract]:The new duck reovirus disease is a new epidemic disease in recent years. It is characterized by irregular hemorrhage of liver, spleen, necrosis and myocardium, hemorrhage of bursa of cavity, etc. It was originally called "duck white spot disease", "duck new liver disease", "duck necrotic hepatitis disease" and so on. It has been established that the disease is caused by a new duck reovirus. In order to improve the accuracy, sensitivity and effectiveness of the diagnosis of novel duck reovirus infection, a method of NDRV fluorescence quantitative RT-PCR detection and RT-LAMP detection was established in this study. In addition, the adaptability of the virus to different cells was analyzed, and the virus was cloned, purified and subcultured on chicken embryo fibroblast (CEF), which laid a foundation for the development of attenuated virus vaccine. This study is divided into three parts: the establishment of 1.NDRV fluorescence quantitative RT-PCR detection method according to the sequence of NDRV S1 gene designed a pair of specific primers to establish a real-time quantitative RT-PCR detection method based on SYBR Green 鈪，
本文编号：2299161
[Abstract]:The new duck reovirus disease is a new epidemic disease in recent years. It is characterized by irregular hemorrhage of liver, spleen, necrosis and myocardium, hemorrhage of bursa of cavity, etc. It was originally called "duck white spot disease", "duck new liver disease", "duck necrotic hepatitis disease" and so on. It has been established that the disease is caused by a new duck reovirus. In order to improve the accuracy, sensitivity and effectiveness of the diagnosis of novel duck reovirus infection, a method of NDRV fluorescence quantitative RT-PCR detection and RT-LAMP detection was established in this study. In addition, the adaptability of the virus to different cells was analyzed, and the virus was cloned, purified and subcultured on chicken embryo fibroblast (CEF), which laid a foundation for the development of attenuated virus vaccine. This study is divided into three parts: the establishment of 1.NDRV fluorescence quantitative RT-PCR detection method according to the sequence of NDRV S1 gene designed a pair of specific primers to establish a real-time quantitative RT-PCR detection method based on SYBR Green 鈪，
本文编号：2299161
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