畜牧兽医学报2011，Vol.42Issue(7)：1039-1045,7.
免疫PCR检测微量H5亚型禽流感病毒
Immuno-PCR Assay for Detection of H5 Hemagglutinin Subtype Avian Influenza Virus
邓明俊 1肖西志 2吴振兴 1张彦明 2辛学谦 2郑小龙 1王群 2王昌军2
作者信息
- 1. 西北农林科技大学动物医学院,杨凌712100
- 2. 山东出入境检验检疫局,青岛266002
- 折叠
摘要
Abstract
In order to detect the low concentration of avian influenza virus (AIV) in the tracheal and cloacal swabs taken from the HPAI-infected poultry, an indirect immuno-polymerase chain reaction (immuno-PCR) assay and an indirect sandwich immuno-PCR assay, based on Immunology and polymerase chain reaction (IPCR) which combining enzyme immunoassay with DNA amplification, were developed. For the indirect immuno-PCR assay, TopYield microtiter plates were coated with purified AIV/H5 protein that was recognized by anti-AIV/H5 monoclonal antibody. For the indirect sandwich immuno-PCR assay, the monoclonal antibody was immobilized on TopYield plates for detecting H5 protein that was recognized by its polyclonal antibodies. Reporter DNA was prepared by PCR amplification using biotinylated 5-primers, and it was coupled with biotinylated antibodies through streptavidin. In order to increase sensitivity and reduce background noise, the amounts of reporter DNA and streptavidin were optimized. Using the optimized concentration of reporter DNA and streptavidin, both indirect and indirect sandwich immuno-PCR assays detected AIV/H5 protein as low as 1 fg. These results are a nearly 1000-fold improvement over conventional indirect ELISA and indirect sandwich ELISA methods.Our data demon-strate that this Mab-based immuno-PCR method provided a platform capable of mass screening of clinical samples for AIV H5 subtype and could serve as a model for other immuno-PCR assays.关键词
免疫PCR/禽流感病毒/H5血凝素蛋白Key words
immuno-PCR/ avian influenza virus/ H5 hemagglutinin protein分类
农业科技引用本文复制引用
邓明俊,肖西志,吴振兴,张彦明,辛学谦,郑小龙,王群,王昌军..免疫PCR检测微量H5亚型禽流感病毒[J].畜牧兽医学报,2011,42(7):1039-1045,7.
