以金磁微球为载体的H5N1 AIV免疫PCR检测方法的建立

Immuno-PCR for detection of H5N1 avian influenza virus using magnetic gold particles as carriers

【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(Avianinfluenzavirus,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AIVH5N1血凝素蛋白的检测抗体分子连接,H5N1AIV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H5N1AIV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1ng/mL,最适链亲和素工作质量浓度为20ng/mL,该方法可成功检测到10-4EID50/mL的H5N1AIV,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H5N1AIV的方法。

[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 10^2-10^-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.