作　　者： ; ; ; ; ; ; ; ; ; ;

机构地区： 扬州大学兽医学院

出　　处： 《中国兽医学报》 2009年第12期1534-1538,1556,共6页

摘　　要： 从GenBank下载禽流感病毒(Auian in fluenza virus,AIV)NP基因序列,通过比对选取NP基因中较为保守的片段,设计1对引物,通过RT-PCR方法扩增270 bp的目的片段,经测序确认目的片段序列正确后,用地高辛标记扩增产物制备探针。在BSL-3实验室,用AIV人工感染鸡胚成纤维细胞和SPF鸡后,制作细胞涂片和组织石蜡切片,然后在经前处理后的细胞涂片和石蜡切片上进行原位RT-PCR,再与地高辛标记的探针进行原位杂交,对细胞和组织中的AIV进行检测和定位。研究结果表明,本方法能检测出约1μg/L质粒的DNA,并能特异性地在细胞涂片和石蜡组织切片中检测和定位AIV,且成纤维细胞感染病毒8 h后即可检测到阳性信号,具有良好的特异性和较高的敏感性,为动物组织中AIV的检测定位和致病机理研究提供了敏感、特异和更为直观的方法。 Based on the conserved AIV NP gene downloaded from GenBank, we designed a pair of primers and obtained 270 bp segment of NP gene by RT-PCR. After the sequence of RT-PCR segment attested to be correct,the probe was prepared with the RT-PCR segment labeled by DIG. AIV infected CEF sections and paraffin-embedded tissue sections were prepared in BSL-3. RT-PCR was processed on the sections after a series of processing of fixation and protease K digestion. To detect and locate AIV in infected culture cells and tissues, prepared sections and DIG la- beled probe were subjected to specific hybridization, result showed that this method can accurately detect 1 μg/L plasmid DNA and and locate AIV in culture cell sections and paraffin-embedded tissue sections, positive hybridization signal can be detected in the CEF just after 8 h inoculation. Our research developed a more observational and practicable method for AIV group detection, localization and nosogenesis research in animal tissue, with high sensitivity and specificity.

关 键 词： 禽流感 群特异性 细胞涂片 石蜡切片 间接原位

领　　域： [农业科学] [农业科学] [农业科学]