| 摘要: |
| 【目的】将高特异性的抗原-抗体反应与高灵敏性的PCR扩增技术相结合，建立快速检测低浓度H5N1禽流感病毒(Avian influenza virus，AIV)的免疫PCR方法。【方法】以pUC19为模板，采用5′端标记有生物素的引物进行PCR扩增，制备含有生物素的报告DNA分子。以金磁微球为固相吸附载体，通过亲和素-生物素的桥联作用，将含有生物素的报告DNA分子与生物素标记的抗AIV H5N1血凝素蛋白的检测抗体分子连接，H5N1 AIV与检测抗体结合后，体外扩增报告DNA，间接放大低含量病毒信号，建立可有效检测微量H5N1 AIV的免疫PCR方法。对建立的免疫PCR方法的最适报告DNA浓度、最适链亲和素工作浓度、灵敏度和特异性进行确定和评价。【结果】 建立的免疫PCR方法最适报告DNA质量浓度为1 ng/mL，最适链亲和素工作质量浓度为20 ng/mL，该方法可成功检测到10-4 EID50/mL的H5N1 AIV，而且特异性良好。【结论】建立的以金磁微球为吸附载体的免疫PCR是一种灵敏度高、特异性好的检测微量H5N1 AIV的方法。 |
| 关键词: 免疫PCR H5N1禽流感病毒 金磁微球 |
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| 基金项目:山东出入境检验检疫局科技项目（SK200601） |
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| Immuno PCR for detection of H5N1 avian influenza virus using magnetic gold particles as carriers |
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| Abstract: |
| 【Objective】The study was done to detect avian influenza virus (AIV) at low concentrations from tracheal and cloacal swabs of avian influenza infected poultry using a highly sensitive immunological polymerase chain reaction (immuno-PCR) method.【Method】Magnetic gold particles were pre-coated with a capture antibody,a monoclonal anti-AIV H5 Hemagglutinin,and viruses serially diluted ten fold from 102-10-5 EID50/mL.A biotinylated detection antibody bound to the viral antigen was then linked via a streptavidin bridge to biotinylated reporter DNA.After extensive washing,reporter DNA was released by denaturation,transferred to PCR-tubes,amplified,electrophoresed and visualized.To further evaluate the specificity of this IPCR assay for H5N1 AIV,the tracheal swab specimens,taken from chickens which were infected with NDV,H9 subtpye AIV,H7 subtype AIV,IBDV,IBV/H120,were detected by this IPCR.【Result】An optimized immuno-PCR method was able to detect as little as 10-4 EID50/mL H5N1 AIV using an optimal 1 ng/mL of reporter DNA and 20 ng/mL of streptavidin.【Conclusion】Our data demonstrated that this monoclonal antibody based immuno PCR method provides a platform capable of rapid screening of clinical samples for trace levels of AIV H5,and can serve as a model for other immuno PCR assays. |
| Key words: Immuno-PCR H5 subtpye avian influenza virus magnetic gold particles |
