【作者】 贾晓庆；

【导师】 雷治海； 张常印；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2008， 硕士

【摘要】 马流感（Equine influenza,EI）是由马流感病毒引起马的一种急性高度接触传染性、群发性呼吸道疾病,类似于人流感,典型的临床症状为突发高热、咳嗽、呼吸困难、食欲减退。马流感病毒（Equine influenza virus,EIV）属正粘病毒科A型流感病毒,有H7N7和H3N8两个亚型。前者于1979年最后一次在意大利流行后再未分离到,然而,H3N8每年都有发生。目前,马流感没有有效的治疗措施,因此,建立有效的检测方法来监控马流感是非常重要的。1.马流感病毒间接免疫荧光检测方法的建立马流感病毒接种鸡胚,收获的尿囊液经差速离心后,再经蔗糖超速离心,提纯EIV。提纯后的EIV经灭活并与弗氏完全佐剂或弗氏不完全佐剂混和后免疫新西兰兔三次,制备了多克隆抗体,建立了间接免疫荧光试验。其最适工作条件为:80%丙酮4℃固定30 min;一抗最佳浓度为1:80,37℃作用60 min;二抗最佳浓度为1:40,37℃作用60min。本方法可用于EI的检测。2.马流感病毒RT-PCR检测方法的建立从GenBank上调取马H3N8流感病毒HA和NA的基因序列,经过分析,设计并合成两套RT-PcR引物,分别对马流感病毒尿囊液进行扩增,结果能扩增出与目的片断大小一致的条带,胶回收PCR产物进行序列测定,证实为EIV HA和NA的基因。从而建立了RT-PCR检测方法,经各反应条件优化后,发现两套RT-PCR引物均能扩增10 TCID₅₀的尿囊液病毒,对AIV（H5、H7、H9）、H3N1、H3N2、Pi等6种病毒核酸进行扩增,均未扩增出特异性条带,表明它具有良好的特异性,上述结果显示,本文建立的RT-PCR检测方法具有高效、快速、特异、灵敏的特点,可用于EIV的检测和监控。3.马流感病毒实时荧光RT-PCR检测方法的建立从GenBank上调取马H3N8流感病毒的基因序列,针对NA基因相对保守区域的核苷酸序列,用DNAStar和Primer Expression 3.0软件设计1对引物和TaqMan探针,以马流感病毒的cDNA为模板,建立了马流感病毒实时荧光RT-PCR检测方法,经反应条件优化后,对AIV（H5、H7、H9）、H3N1、H3N2、Pi等6种病毒核酸进行扩增,发现具有良好的特异性;对10倍倍比稀释的RNA、cDNA和目的片段的双链DNA进行扩增检测其灵敏度,发现建立的荧光RT-PCR分别能检测0.1 TCID₅₀和20个拷贝的双链DNA,结果显示该方法具有高效、快速、特异、灵敏的特点,可用于马流感病毒的检测和监控。更多还原

【Abstract】 Equine influenza （EI） is a severe acute upper respiratory infection of the horse, just as with human influenza, and typical symptoms include pyrexia, dyspnoea, anorexia and coughing. Spread of equine influenza virus is very rapid and explosive. Equine influenza virus （EIV） is a type A influenza virus, a member of family Orthomyxoviridae, and there are two distinct subtypes, H7N7 and H3N8. It is generally accepted that the former has been no longer existence in the world the last outbreak caused by this virus was in 1979 in Italy, however, outbreaks of EIVcaused by H3N8 viruses occur annually. Currently, there are no effective therapies against EIV infection. Therefore, it is important to establish detection methods to detect and monitor EIV.1. Establishment of indirect immunofluorescent assay for EIVIn this study, EIV was purified and concentrated from infected allantoic fluid of embryonic egg by centrifugation and ultracentrifugation. Hyper-immunized rabbit serum was prepared by immunizing rabbit for three times with inactive purified EIV mixed with Freund’s adjuvant or incomplete Freund’s adjuvant. Then, the indirect immunofluorescent assay （IFA） was established for EIV. The optimal conditions for IFA were determined as follows: EIV was fixed by 80% acetone 30 min at 4℃, anti-sera was diluted for 1:80 and the FITC-labeled antibody was diluted for 1:40. The optimal reactive time of the detecting antibody and conjugate were done at 37℃for 60 min. The IFA established here was sensitive and can be used for detecting EIV.2. RT-PCR to detect the equine influenza virusTwo pairs of RT-PCR primers for HA and NA genes were designed and synthesized based on the nucleotide sequences of the equine H3N8 influenza virus in GenBank. After optimization of the detection methods, they were used to amplify EIV virus allantoic fluid. The result showed that all primers could get the specific bands. Then the two fragments from the RT-PCR products were extracted by Agarose Gel DNA Extraction Kit, respectively, sequenced and confirmed to be EIV gene. Sensitivity to detection of highly purified influenza virus by RT-PCR reached approximately 10 TCID₅₀. The high specificity of the methods was demonstrated by detecting 6 viruses, such as H3N1 virus, Pi virus, H3N2 virus, and so on. Therefore, two RT-PCR methods developed here for EIV detection had the characteristic of high efficiency, speediness, specificity and sensitivity. They can be used to detect and monitor EIV.3. Real time fluorescence RT-PCR to detect the equine influenza virusOne pairs of RT-PCR primers and probes for NA gene were designed and synthesized by DNAStar and Primer Expression 3.0 based on the nucleotide sequences of EIV （H3N8） in GenBank. After optimization of the detection methods, NA real-time fluorescence RT-PCR techniques were developed to amplify EIV. The high specificity of the methods was demonstrated by detecting 6 viruses, such as AIV （H5, H7, H9）, H3N1, H3N2, Pi and SO on. The minimal detection limits of NA real-time RT-PCR were approximately 0.1 TCID₅₀ and 20 copies of double-stranded DNAs. Therefore, NA real-time fluorescence RT-PCR on the basis of the TaqMan technique developed here for EIV detection had the characteristic of high efficiency, speediness, specificity and sensitivity, which could be used to detect and monitor EIV.更多还原