H3亚型犬流感病毒的荧光定量PCR方法建立及其增殖特性研究  

Establishment of a fluorescence quantitative method for detection of H3 subtype canine influenza virus and study on the proliferation characteristics of the method

作　　者：程慧敏 黄程 林健[1] 王小蕾[1] 杨志远[1] 段会娟[1] 赵际成 潘洁[1] 刘月焕[1] 刘立新[1] CHENG Huimin;HUANG Cheng;LIN Jian;WANG Xiaolei;YANG Zhiyuan;DUAN Huijuan;ZHAO Jicheng;PAN Jie;LIU Yuehuan;LIU Lixin(Institute of Animal Husbandry and Veterinary Medicine,Beijing Municipal Academy of Agricultural and Forestry Sciences,Beijing 100097,China)

机构地区：[1]北京市农林科学院畜牧兽医研究所,北京100097

出　　处：《畜牧与兽医》2022年第2期112-118,共7页Animal Husbandry & Veterinary Medicine

基　　金：北京市农林科学院青年基金项目(QNJJ202025)。

摘　　要：旨在建立H3亚型犬流感病毒(canine influenza virus, CIV)荧光定量PCR方法,利用该方法对病毒在犬肾细胞(MDCK细胞)上增殖特性进行研究。根据CIV的血凝素(HA)片段上的HA2基因保守区设计并合成特异性引物,构建CIV-HA重组质粒作为标准品,经过条件优化,建立H3亚型CIV荧光定量检测方法,对其敏感性、特异性和重复性进行评价,并将该方法应用于CIV在MDCK细胞上增殖特性的研究,与病毒滴度(TCID;)进行相关性分析。结果显示:该方法具有较好的敏感性、特异性和重复性,最低检测线可以达到20.8 copies/μL,与犬瘟热病毒、犬细小病毒、犬副流感病毒、犬腺病毒2型、H1亚型流感病毒不发生交叉反应,批间、批内重复性良好(变异系数<1.1%)。将该方法应用于检测CIV在MDCK细胞上的增殖特性,与细胞培养法测定病毒滴度相比,二者具有较好的相关性。本研究初步建立了H3亚型CIV荧光定量检测方法,该方法可为CIV的增殖特性研究提供工具。A real-time PCR method for detecting H3 subtype canine influenza virus(CIV) was established to study the proliferation characteristics of the virus on MDCK cells. Specific primers were designed and synthesized according to the conserved region of the HA2 gene of the civ-ha fragment, and civ-ha recombinant plasmids were constructed as the standard. After optimizing the conditions, the fluorescence quantitative detection method for detecting H3 subtype canine influenza virus was established;and its sensitivity, specificity and repeatability were evaluated. The method was then applied to the study of the proliferation characteristics of CIV on MDCK cells, and for analysis with the virus titer(TCID;). The results showed that the method established here had good sensitivity, specificity and repeatability. The minimum detection line reached 20.8 copies/μL. There was no cross reaction with canine distemper virus, canine parvovirus, canine parainfluenza virus, canine adenovirus(type 2) and influenza virus H1 subtype, and the repeatability between batches and within batches was good(CV<1.1%). In comparison, the present method and the cell culture method had a certain correlation. In conclusion, in this study, a fluorescence quantitative method for detecting H3 subtype canine influenza virus(CIV) was successfully established. This method could serve as a tool for research on and production of CIV.

关 键 词：犬流感病毒 H3亚型 荧光定量PCR 染料法 增殖特性 

分 类 号：S831.7[农业科学—畜牧学]