中国动物传染病学报Issue(1)：16-24,9.
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
摘要
Abstract
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/荧光定量PCRKey words
H3 subtype/H9 subtype/Avian influenza virus/fluorescent quantitative PCR分类
农业科技引用本文复制引用
申伟霞,李泽君,田巧珍,陈圆,胡涛,闫丽萍,李国新,李雪松,滕巧泱,赵宇军..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[J].中国动物传染病学报,2014,(1):16-24,9.基金项目
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 （）
