H5和H9亚型禽流感病毒反向斑点杂交同步检测法的建立  

Development of reverse dot blot hybridization for simultaneous detection of subtype H5 and H9 avian influenza virus

作　　者：王伟[1,2] 薛强[2] 邹明强[2] 柴同杰[1] 田甜[2] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018 [2]中国检验检疫科学研究院,北京100123

出　　处：《西北农林科技大学学报（自然科学版）》2010年第4期7-13,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国际科技合作项目"便携式微流体PCR分析仪研制及检验检疫应用"(2009DFA32720);国家质检总局科技计划项目(2007IK184)

摘　　要：【目的】建立H5和H9亚型禽流感病毒的反向斑点杂交鉴别诊断方法。【方法】在H5和H9亚型禽流感病毒血凝素基因的保守区段内,选择设计2对引物,RT-PCR扩增后将目的片段克隆到pGEM-T载体中,得到重组质粒pGEM-T-H5与pGEM-T-H9。以重组质粒为模版,选择合成寡核苷酸探针与生物素标记引物。将探针通过紫外交联方法固定在带正电荷的尼龙膜上,用生物素标记引物扩增特异性片段,扩增产物经热变性后与探针进行杂交,杂交后显色,出现明显蓝紫色斑点的判定为阳性,无斑点判定为阴性。【结果】利用建立的反向斑点杂交方法检测,H5、H9亚型禽流感病毒的血凝效价分别可达到2-9.6和2-11.9,杂交特异性好,且2亚型间无交叉反应。【结论】建立了H5和H9亚型禽流感病毒的反向斑点杂交检测方法,该方法灵敏度高、特异性好,可以实现H5与H9亚型禽流感病毒的鉴别诊断。[Objective] The study was to develop a reverse dot blot hybridization technology for avian influenza virus （AIV） H5 and H9 subtype identification. [Method] Two pairs of primers were designed according to the conserved sequences of hemagglutinin genes. The interested fragments were amplified and cloned into a vector pGEM-T. The recombinant plasmids were named pGEM-T-H5 and pGEM-T-H9 respectively. Based on the recombinant plasmid, oligonucleotide probes and biotinylated primers were designed. The oligonucleotide probes were immobilized onto a positively charged nylon membrane by UV radiation. Taget DNA fragments were amplified with biotinylated primers and then hybridized with oligonu- cleotide probes on the membrane. Color reaction was carried out after hybridization. The presence of clearly visible purple-blue spots on the membrane was considered to be positive hybridization reaction. [Result] 2 96 hemagglutination titers of H5 subtype virus could be detected by reverse dot blot hybridization assay,and the detection limit of H9 subtype was 2^-11.9 hemagglutination titers. No cross-hybridization was detectecd between subtype H5 and Hg. [Conclusion] Reverse dot blot hybridization assay for identification of AIV subtype H5 and H9 was specific and sensitive,which might become a rapid,effective method for the identification of AIV.

关 键 词：反向斑点杂交 H5 H9亚型禽流感病毒 血凝素 灵敏性 稳定性 

分 类 号：S855.3[农业科学—临床兽医学]