| 摘要: |
| 【目的】 建立H5和H9亚型禽流感病毒的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。 |
| 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 |
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| 基金项目: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） |
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| Development of reverse dot blot hybridization for simultaneous detection of subtype H5 and H9 avian influenza virus |
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| Abstract: |
| 【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 oligonucleotide 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】 2-9.6 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 H9.【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. |
| Key words: reverse dot blot hybridization avian influenza virus subtype H5 and H9 hemagglutinin sensitivity stability |
