用原位RT-PCR对BHK-21细胞中的FMDV检测和定位

目的为了明确FMDV在BHK-21细胞中的复制和增殖部位,建立原位检测FMDV的方法。方法以地高辛标记的寡核苷酸作为探针,用间接原位RT-PCR技术对实验接种FMDV的BHK-21细胞中的FMDV RNA进行检测和定位。结果在接毒细胞的细胞质中有大量强阳性蓝染颗粒,阴性对照细胞中没有蓝染颗粒出现,也没有明显的背景染色。结论结果表明FMDV RNA在细胞的细胞质中进行复制和增殖,同时也表明原位RT-PCR是检测细胞内病毒的一种敏感、特异的方法。

Detection and localization of the foot and mouth disease virus in BHK-21 cells by RT-PCR in situ

In order to localize the duplication and reproduction of Foot and Mouth Disease(FMD) virus(FMDV) in BHK-21 cells and to establish a method of detecting FMDV with hybridization in situ,oligonucleotide probes were labeled with digoxigenin-11-dUTP at 3'end,and the localization of FMDV in HEK21 cells experimentally infected with FMDV O/Akesu/58 strain(10~7 TCID_(50)) was determined by RT-PCR in situ.The experimental results showed that large amount of strongly positive blue staining granules was found in the cytoplasm of infection cells 4 hours after inoculation of viruses,while no such blue staining granule was observed in the un-infected control cells and without obvious background staining.So,it is strongly suggested that reproduction and duplication of FMDV in the cytoplasm of infection cells occur,and it is proved that the RT-PCR in situ is a sensitive method to be used for the detection and localization of FMDV in infection cells.