【摘要】：新型鴨呼腸孤病毒病是近年來(lái)新出現(xiàn)的疫病,主要特征為肝、脾臟不規(guī)則出血、壞死與心肌、腔上囊出血,最初被稱為“鴨白點(diǎn)病”、“鴨新肝病”、“鴨壞死性肝炎病”等�，F(xiàn)已確定該病是由新型鴨呼腸孤病毒引起。為了提高新型鴨呼腸孤病毒感染診斷的準(zhǔn)確性、靈敏度和實(shí)效性,本研究建立了NDRV熒光定量RT-PCR檢測(cè)方法和RT-LAMP檢測(cè)方法。另外,本研究還對(duì)該病毒在不同細(xì)胞上的適應(yīng)性進(jìn)行分析,并將該病毒在雞胚成纖維細(xì)胞(CEF)上進(jìn)行了克隆純化與傳代,為該病弱毒疫苗的研發(fā)奠定了基礎(chǔ)。本研究分為三部分:1.NDRV熒光定量RT-PCR檢測(cè)方法的建立根據(jù)NDRV S1基因序列設(shè)計(jì)了1對(duì)特異性引物,建立基于SYBR GreenⅠ的實(shí)時(shí)熒光定量RT-PCR檢測(cè)方法。根據(jù)含目的基因的質(zhì)�？截悢�(shù)與定量反應(yīng)Ct值的關(guān)系,繪制了標(biāo)準(zhǔn)曲線。該方法敏感性高,比普通常規(guī)PCR高100倍,并具有良好的特異性和重復(fù)性。利用該方法對(duì)人工感染死亡雛鴨的不同組織器官進(jìn)行檢測(cè),結(jié)果發(fā)現(xiàn)脾臟和肝臟中的含毒量較高。2.NDRV RT-LAMP檢測(cè)方法的建立基于NDRV S3基因的6個(gè)保守區(qū)域設(shè)計(jì)了4條LAMP引物,利用Bst DNA聚合酶在63℃恒溫保持45min即可完成反轉(zhuǎn)錄和擴(kuò)增反應(yīng),由此建立了RT-LAMP檢測(cè)方法。該方法具有良好的特異性,敏感性為常規(guī)RT-PCR方法的100倍。臨床應(yīng)用結(jié)果表明,該方法與病毒分離鑒定方法的符合率為98%,而且對(duì)儀器要求低,適于基層實(shí)驗(yàn)室和現(xiàn)場(chǎng)檢測(cè)。3.NDRV細(xì)胞適應(yīng)性分析將NDRV分別在DEF、CEF、Vero、BHK-21、DF-1等細(xì)胞傳代至適應(yīng)穩(wěn)定后,分別收集不同時(shí)間段的病毒液并測(cè)定其TCID50,繪制病毒的生長(zhǎng)曲線,篩選出鴨新型呼腸孤病毒最適應(yīng)的細(xì)胞為CEF細(xì)胞。將NDRV在CEF進(jìn)行克隆純化,并進(jìn)行了傳代,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，

本文編號(hào)：2299161