CIAV及FAdV检测方法的建立及其在弱毒疫苗污染检测中的应用

发布时间：2018-02-25 17:10

  本文关键词： 鸡传染性贫血病毒 禽腺病毒 弱毒疫苗 SYBR GreenⅠ荧光定量PCR PCR 污染　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：鸡传染性贫血病(Chicken infectious anemia,CIV)是由鸡传染性贫血病毒(Chicken infectious anemia virus,CIAV)引起的雏鸡发生再生障碍性贫血,以全身性淋巴组织萎缩为特点的免疫抑制性传染病。禽腺病毒(Fowl adenovirus,FAdV)可引发鸡产生包涵体肝炎(IBH)、心包积液综合征(HPS)和肌胃糜烂(GE)等疾病。CIAV和FAdV均可造成感染鸡群处于免疫抑制状态,使鸡群对其他病原易感性增强,同时对马立克、传染性法氏囊病及禽网状内皮增生病等疾病的疫苗保护力降低,从而导致鸡群在共感染或继发感染等其他病原时死亡率升高,对肉鸡产业和SPF鸡蛋的生产造成严重的经济损失。CIAV和FAdV除垂直传播和水平传播外,同时还可以通过疫苗污染途径传播,因此本研究建立了用于疫苗中监测CIAV和FAdV的快速检测方法,将有助于企业能够准确而快速地检测禽弱毒疫苗中的CIAV和FAdV污染。一、CIAV SYBR Green检测方法的建立及其在疫苗污染检测中的应用根据CIAV基因组保守区域VP2部分基因设计合成1对扩增片段大小为154bp的特异性引物,同时通过优化反应条件建立了快速检测CIAV的SYBR GreenⅠ荧光定量PCR方法,进行敏感性、特异性和重复性试验。结果显示,荧光定量法检测CIAV的Ct阈值与标准品浓度在6.36×107拷贝/μL-6.36拷贝/μL之间呈现良好的线性关系,相关系数R2=0.999,斜率为-3.186,灵敏度比常规PCR高1000倍。该方法与禽网状内皮组织增生病病毒(REV)、禽白血病病毒(ALV)、马立克氏病病毒(MDV)等均无交叉反应,特异性良好;批内和批间重复试验显示变异系数均小于2.5%,呈现良好的可重复性。在模拟试验中,该方法能够检测到1000羽份弱毒疫苗中1个EID50的低剂量污染,应用该方法从送检的14种(批)商品化禽用弱毒疫苗中检测到2份为CIAV阳性,阳性样品按照经典的SPF鸡检查法进行检测也为CIAV阳性。综合上述结果表明,本研究建立的SYBR GreenⅠ荧光定量PCR检测方法灵敏度高,特异性强,重复性好,适合用于疫苗中CIAV污染的检验。二、FAdV PCR检测方法的建立及其在疫苗污染检测中的应用根据GeneBank上已发表FAdV的JSJ13分离株hexon基因,设计合成了一对扩增片段大小为1310bp的特异性引物,通过优化反应条件建立PCR检测方法,并利用该方法对32批弱毒活疫苗进行检测。结果显示,其中一支La Sota株的新城疫活疫苗(Clone30)FAdV呈阳性。通过鸡胚接种对疫苗中FAdV进行分离鉴定,利用建立的PCR法扩增,结果显示出与疫苗检测相同的hexon基因条带,片段长度为1310bp,证明该新城疫活疫苗中存在FAdV污染并对其成功实现了分离鉴定。本分离株(FAdV-N22)与GenBank上已发表的其他不同参考株hexon基因的同源性进行比较分析并做进化树发现,FAdV-N22株与其他FAdV毒株hexon基因核苷酸序列的同源性介于98.3%-99.8%之间,其中与2015年中国分离株JSJ13株(FAdV-4型)同源性最高,为99.8%;相比之下,与A、B、D和E种同源性较低,与II群和III群的同源性更低,同源性分别仅有56.9%和40.9%;进化树显示所有毒株分成两大组,组Ⅰ包含A-E五个种,组Ⅱ只有属于FAdV-II亚群的出血性肠炎病毒(HEV)和属于FAdV-III亚群的产蛋下降综合征病毒(EDSV)两个株,由此证明,FAdV-N22分离株属于禽腺病毒C种的FAdV-4型。将FAdV-N22分离株进行动物致病性试验,结果表明,鸡接种FAdV-N22后,死亡率为100%,对照组健活。1d龄攻毒后1d全部死亡,无症状表现;3w龄攻毒后,3d内无明显症状,第4d开始出现大量死亡,病鸡精神萎靡、羽毛蓬松,肛拭子PCR检测全部为FAdV阳性。解剖鸡出现心包积液等典型临床症状;病理学组织变化显示肝脏脂肪变性、出血,肝细胞核内可见嗜碱性包涵体。本研究建立了FAdV的PCR检测方法,利用这一方法能够快速有效的检测弱毒疫苗中FAdV的污染,有利于对弱毒活疫苗中FAdV等家禽外源病毒污染的监测。利用此方法并结合病毒分离鉴定,在我国家禽活疫苗中首次分离鉴定到FAdV-4型,这些结果表明活疫苗中FAdV的污染可能是FAdV感染鸡群并发病的重要途径之一。
[Abstract]:Chicken infectious anemia (Chicken infectious, anemia, CIV) is composed of chicken infectious anemia virus (Chicken infectious anemia virus, CIAV) caused by the occurrence of chicken aplastic anemia, with systemic lymphoid tissue atrophy of immune suppression diseases. Avian adenovirus (Fowl adenovirus, FAdV) can cause chickens to produce inclusion B (IBH), pericardial effusion syndrome (HPS) and gizzard erosion (GE) and FAdV.CIAV and other diseases can be caused by infection of chickens in immunosuppressed chickens, the enhancement of susceptibility to other pathogens, at the same time to Marek, the vaccine protection of infectious bursal disease virus and Reticuloendotheliosis disease decreased, resulting in chickens in the total infection or secondary infection of other pathogens such as increase in mortality, causing serious economic losses of.CIAV and FAdV in the vertical and horizontal transmission of SPF and egg production of broiler industry, At the same time can also be spread through contaminated vaccine approaches, this study established a fast detecting method for monitoring CIAV and FAdV vaccine, will help enterprises to quickly and accurately detect avian vaccine in CIAV and FAdV pollution. A Green method for detection of CIAV, SYBR and built in vaccine contamination detection according to the application of CIAV genome conserved regions of VP2 gene were designed and synthesized 1 pairs of specific primers amplified fragment size was 154bp, at the same time through the optimization of reaction conditions for the establishment of a SYBR Green 1 fluorescent quantitative PCR method for rapid detection of CIAV, sensitivity, specificity and repeatability. The results show that the Ct threshold and standard concentration detection the fluorescent quantitative CIAV method in 6.36 * 107 copies / L-6.36 copies / L showed a good linear relationship, the correlation coefficient R2=0.999, the slope is -3.186, sensitivity is 1000 times higher than the conventional PCR. With the method of reticuloendotheliosis virus (REV), avian leukosis virus (ALV), Marek's disease virus (MDV) there were no cross reactions, good specificity; intra and inter repeat test showed that the coefficient of variation was less than 2.5%, showing a good repeatability. In the simulation test, the method able to detect 1000 samples of low dose pollution 1 EID50 attenuated vaccine, from 14 for the application of this method (Group) commercial poultry attenuated vaccine was detected in 2 cases were CIAV positive, the positive samples in accordance with the law of SPF chickens were detected for the inspection of the classic CIAV positive. The above results show that the sensitivity of SYBR Green 1 fluorescent quantitative PCR detection method established in this study, strong specificity, good repeatability, suitable for the inspection of CIAV pollution in the vaccine. In two, the establishment of FAdV method for the detection of PCR and its application in vaccine root pollution detection in GeneBank according to the published FAdV The JSJ13 isolates hexon gene, the amplified fragment size was 1310bp with specific primers were designed and synthesized by optimizing the reaction conditions of the PCR detection method, and to detect the 32 batch of live attenuated vaccine by using this method. The results showed that one La Sota strain of Newcastle disease vaccine (Clone30) was FAdV positive. By isolation and identification of the vaccine in FAdV chicken embryo inoculation, amplified by the PCR method, the results show that the hexon gene with the same vaccine detection zone, the fragment length of 1310bp, the Newcastle disease live vaccine FAdV pollution and its successful implementation. The isolation and identification of isolates (FAdV-N22) were comparative analysis and phylogenetic tree showed that the homology with GenBank has been published on the other reference strains of hexon gene between FAdV-N22 strain and other strains of FAdV hexon gene nucleotide sequence homology between 98.3%-99.8%, which 涓，

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