H5N1禽流感病毒微磁粒捕捉系统和SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立  

Development of magnetic bead capture system and SYBR Green Ⅰ real time RT-PCR technique for detection of avian influenza A (H5N1) virus

作　　者：张坤[1,2] 李刚[1,3] 贾凤芹[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100093 [2]西南大学动物科技学院,重庆400715 [3]动物营养学国家重点实验室,北京100093

出　　处：《中国兽医学报》2009年第9期1144-1148,共5页Chinese Journal of Veterinary Science

基　　金：联合国FAO/IAEA资助项目(14133)

摘　　要：利用禽流感H1N1抗体包被微磁珠制备免疫磁珠,再通过抗原抗体反应得到了含有N1亚型的病毒,然后设计针对H5亚型的特异性引物,同时构建含有H5亚型HA基因部分序列的标准质粒,定量后作为模板,建立了可以检测H5亚型禽流感病毒的SYBR GreenⅠ荧光定量RT-PCR(RRT-PCR)方法。结果表明,制备的免疫磁珠可以吸附4个血凝单位的H5N1流感病毒和5个血凝单位的H1N1流感病毒,而对H9N2亚型流感病毒无特异性结合,从而可以富集N1亚型的流感病毒。H5亚型RRT-PCR检测灵敏度可达到4.6拷贝/μL,线性范围达5个数量级;特异性强,与NDV和H1、H9亚型禽流感病毒不发生交叉反应。因此,利用该方法可以很好地检测H5N1禽流感病毒。The virus are captured using newly established magnetic beads which are coated by anti-H1N1 antibodies. Using this method, the virus was concentrated. A pair of primers was designed in subtype H5 AIV HA gene conservative region. Meanwhile,the standard plasmid containing partial sequence of subtype H5 AIV HA gene was constructed as a template,a SYBR Green Ⅰ real time RT PCR was established. The results showed magnetic beads could absorb four HA units of AIV （H5N1） and five HA units of AIV （H1N1）, but did not bind with AIV （H9N2）. Depending on the enrichment of subtype N1 AIV, the RRT PCR for detecting AIV H5 subtype showed the sensitivity was 4.6 copies/μL,while it was linear with 5 log dynamic rang. The specificity assay showed it is of high specificity,with no cross reaction to NDV, H1 and H9 AIV. So this method,magnetic bead capture system and SYBR Green Ⅰ real time RT-PCR could be applicabe to detection of AIV （H5N1）.

关 键 词：H5N1禽流感病毒 微磁粒捕捉系统 荧光定量RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学] R535[农业科学—兽医学]