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中文题名:

 禽流感病毒和新城疫病毒双重RT-PCR检测及野鸭源禽流感病毒M基因序列分析    

姓名:

 刘婧君    

学号:

 2005107080    

保密级别:

 公开    

论文语种:

 chi    

学科代码:

 090602    

学科名称:

 预防兽医学    

学生类型:

 硕士    

学位:

 兽医硕士    

学校:

 南京农业大学    

院系:

 动物医学院    

专业:

 预防兽医学    

研究方向:

 预防兽医学    

第一导师姓名:

 费荣梅    

第一导师单位:

 南京农业大学动物医学院    

完成日期:

 2008-06-19    

答辩日期:

 2008-06-19    

外文题名:

 DETECTION AND SEQUENCE ANALYSIS OF INFLUENZA A VIRUS AND NEWCASTLE DISEASE VIRUS FROM WILD DUCKS WINGTING IN WETLANDS    

中文关键词:

 禽流感病毒 ; 新城疫病毒 ; 双重RT-PCR ; 野鸭 ; 进化树分析    

外文关键词:

 avian influenza virus ; newcastle disease virus ; duplex RT-PCR ; migratory ducks ; phylogenetic tree analysis    

中文摘要:
1. 本研究根据GenBank中发表的禽流感病毒和新城疫病毒的基因组序列,利用DNAStar软件分别对AIV、NDV的基因序列进行同源性比较,设计筛选出两对双重RT-PCR反应的特异性引物,其中一对是禽流感病毒的通用引物,扩增出大小约为190bp的片段;另一对为新城疫病毒的通用引物,扩增出大小约为363bp片段。分别建立了AIV、NDV的单一RT-PCR检测方法,用琼脂糖凝胶电泳鉴定PCR产物,结果证明所扩增的产物均为特异性的核酸片段。在单一RT-PCR方法的基础上,进行引物浓度、退火温度等反应条件的优化后,建立了AIV和NDV的双重RT-PCR检测方法,可以在一个PCR反应中同时扩增出禽流感病毒和新城疫病毒两种不同大小的特异性片段,并对该反应条件进行了优化。此双重RT-PCR诊断方法的敏感性要高于病毒分离方法,且缩短了检出时间,表明该方法具有高度的特异性和灵敏性,可从分子水平对禽流感病毒和新城疫病毒进行早期快速诊断和流行病学调查。 2. 2007年2月从江苏省盱眙县等地采集了8种野鸭泄殖腔拭子70份,RT-PCR检测结果显示14.29%(10/70)的野鸭带有A型流感病毒,其中斑嘴鸭和赤麻鸭的阳性率最高,为25%,表明江苏省越冬野鸭的流感病毒库比较庞大。通过SPF鸡胚尿囊腔接种分离病毒,得到病毒6株,病毒分离率为8.57%(6/70)。有1.43%(1/70)的野鸭带有新城疫病毒,并通过SPF鸡胚尿囊腔接种分离病毒,得到病毒1株,分离率为1.43%(1/70)。本研究分离的6株流感病毒通过RT-PCR扩增出的NS基因中大小为190bp的一段高度保守的片段,同源性为100%,与猪源的H5N1同源性为97%,在苏格兰分离的H5N1的同源性为100%,河南、山东、湖南、湖北在禽源中分离的H5N1同源性竟高达99%。野鸭可能在A型流感病毒的传播过程中起重要作用。分离的1株新城疫病毒F基因的一段为363bp的保守序列,与弱毒苗株 Lasota、B1同源性为100%,与国内毒株JS/1/05/Go、SD/1/04/Go、ZJ/2000、SD/1/03/Go同源性为100%,均为鹅源。进一步证明了野生水禽在新城疫病毒的进化中有着重要的作用。 3.对2004年12月到2005年3月分离的5株病毒的M基因进行了全长克隆,对M基因核苷酸序列及其两个产物的氨基酸序列与1973~2007年间分离的已发表的41株各种血清亚型的、不同地区不同宿主来源的A型流感病毒的M基因序列用MegAlign(DNAStar)序列同源性分析,用PHYlip软件绘制基因进化树进行分析,结果表明5株病毒M基因进化同源性都在95%以上,同源性高低与野鸭种类、分离地点、分离时间无关,说明野鸭中流感病毒具有多样性。本试验中spdkXY605、spdkXY705、spdkXY905、maXY704特别值得注意,此4株病毒M基因在同一进化分支上,同源性在99.5%以上。与我国国内河北、吉林等省家禽所感染的高致病性禽流感病毒H5N1亲缘关系密切,与在日本北海道鸭中分离的H5N1的亲缘关系较近。在M1、M2基因氨基酸序列比对中,发现此次分离的5株流感病毒与欧亚系流感病毒H9亚型在遗传演化地位上关系密切。可能说明江苏省越冬的野鸭可能在流感病毒传播过程中起重要作用。
外文摘要:
Avian influenza virus (AIV) and Newcastle disease virus(NDV) are pathogens of social and economical importance known to be disseminated throughout the world by migratory birds. To improve the efficacy and the sensitivity of different diagnosis for NDV and AIV, a duplex reverse transcription-polymerase chain reaction (RT-PCR) technique was established in the present study. To do this, two sets of primers were designed according to the sequences of AIV and NDV published in GenBank. One pair of primer was 190bp and the other was 363bp in length, were expected to be amplified, respectively. After the two sets of primers were combined and used for amplification trials, the conditions of PCR for detection of the two viruses were optimized by orthogonal assay. Based on the single RT-PCR assay that detected each of the two viruses respectively, a duplex RT-PCR system for both of them in one tube was established to distinguish the two viruses. The results suggested that the established duplex RT-PCR system was much more sensitive, specific, rapidly and could be applied for early clinical diagnosis of AIV and NDV. Furthermore, it could also be considered to be used on the research of pathogenesis of the two viruses. 70 cloacal swabs from 70 feral ducks including 8 types wintering in Hongze Lake in Jiangsu province collected from February 2007 were checked for the presence of influenza A viruses and Newcastle disease virus using duplex RT-PCR. Results suggest that the prevalence of influenza viruses is up to 14.29%(10/70). The prevalence in Ruddy Shelduck, Spotbilled duck is up to 25%. The results reveal that enormous pools of influenza viruses are present in migratory ducks in these areas. Sequences analysis of NS gene suggest that these viruses maybe a great threat to public and poultry health. The prevalence of Newcastle disease virus is up to 1.43%(1/70). Sequences analysis of F gene showed that the identities with Lasota、B1 strains were high. Analysis of M gene nucleotide sequences and deduced amino acid sequences from five isolates suggest that M gene of spdkXY605、spdkXY705、spdkXY905、maXY704、btHZ1405 are closely related to HPAIV H5N1 from poultry in Hebei、Jilin Province which indicate that migratory ducks wintering in Jiangsu Province maybe involved in long distance transimission of avian influenza viruses .
中图分类号:

 S852.6    

馆藏号:

 2005107080    

开放日期:

 2020-06-30    

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