H3、H9亚型禽流感病毒双重荧光定量PCR方法的建立及初步应用  

DEVELOPMENT AND PRELIMINARY APPLICATION OF FLUORESCENT QUATITATIVE PCR ASSAY FOR DETECTION OF H3 AND H9 SUBTYPES OF AVIAN INFLUENZA VIRUS

作　　者：申伟霞[1,2] 田巧珍[2] 陈圆[2] 胡涛[2] 闫丽萍[2] 李国新[2] 李雪松[2] 滕巧泱[2] 赵宇军[1] 李泽君[2] 

机构地区：[1]山西农业大学动物科技学院,太谷030801 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2014年第1期16-24,共9页Chinese Journal of Animal Infectious Diseases

基　　金：国家高技术研究发展计划(863计划)(2011AA10A200);国家自然科学基金项目(31302115);中央级公益性科研所基本科研业务费专项资金项目(2013JB06);公益性行业(农业)科研专项(201003012);上海市科委现代农业领域重点科技攻关项目(12391902000)

摘　　要：虽然H3、H9亚型禽流感病毒（Avian influenza virus, AIV）是低致病性禽流感病毒，但其具有跨宿主感染哺乳动物的能力，对社会公共卫生安全具有重大的潜在危害，因而对这些亚型病毒的监测极其重要。通过比对GenBank上H3、H9亚型禽流感病毒HA基因序列，设计了分别针对H3 AIV和H9 AIV的特异性探针。特异性试验和标准曲线试验的结果显示，这两个探针仅分别特异性识别H3、H9亚型AIV，且具有较好的扩增效率。通过重组质粒pMD19-T-H3HA和pMD19-T-H9HA 10倍梯度稀释液为模板，进行敏感性实验。结果表明，本研究所建立的荧光定量PCR方法能检测到100个DNA拷贝数H3 AIV和10个DNA拷贝数的H9 AIV，比传统PCR方法敏感性分别提高了100倍和1000倍。此外，本方法还能检测到10 EID50的H3亚型AIV或H9亚型AIV，比传统PCR方法检测敏感性均提高了1000倍。批间和批内试验的变异系数均小于3%，表明本研究建立的方法具有较好的重复性。此外，与传统PCR方法相比，用H3、H9亚型AIV双重荧光定量PCR方法检测人工感染动物的肺组织时，针对H3 AIV的敏感性提高了10~100倍，针对H9 AIV的敏感性提高了100倍。综上所述，本研究所建立的H3、H9亚型禽流感病毒双重荧光定量PCR方法具有较高的特异性和敏感性，对H3、H9亚型禽流感病毒监测具有重要的应用价值。Although H3 and H9 subtypes of Avian influenza virus （AIV） are low pathogenic in birds, they have adapted abilities to infect mammalians, which poses potential threat to human beings. Therefore, surveillance of H3 and H9 subtypes of AIV is important for public health. In the present study, TaqMan primers specific for either H3 or H9 subtype were designed to develop fluorescent quantitative PCR （qPCR） by comparing the homology of HA gene sequences submitted on the GenBank. The specificity testing and amplification trials demonstrated that TaqMan primers only recognized their respective H3 or H9 subtype. The sensitivity testing of the assay was performed using 10 fold serial dilutions of pMD19-T-H3HA and pMD19-T-H9HA as templates. The result showed that qPCR was able to detect 100 DNA copies of H3 subtype or 10 DNA copies of H9 subtype, which was 100 fold or 1000 fold higher than conventional PCR. In addition, this assay was also able to detect 10 EID50 of both H3 and H9 subtypes, which was 1000 fold higher than conventional PCR. The inter-and intra-assay trials demonstrated that the variations were less than 3%for both subtypes, indicating its good reproducibility. Moreover, qPCR was used to detect viruses in lung samples from experimentally infected chickens. As compared to conventional PCR, qPCR was 10-100 fold more sensitive for H3 subtype or 100 fold more sensitive for H9 subtype than conventional PCR. Therefore, qPCR method developed in this study showed high specificity and sensitivity and could be used for epidemiological study of H3 and H9 subtypes.

关 键 词：H3 H9 禽流感病毒 

分 类 号：S852.659.5[农业科学—基础兽医学]