利用荧光定量PCR检测B.ovis侵染巨噬细胞RAW264.7的相对数量

Detection of Comparative Quantity of Brucella ovis during the Infected RAW264.7 Macrophage by Real-Time PCR

为了探讨布鲁氏菌胞内寄生的机制,采用荧光定量PCR方法对进入巨噬细胞的布鲁氏菌的数量进行检测,克隆了绵羊种布鲁氏菌019株的16SrRNA基因和小鼠巨噬细胞RAW264.7的GAPDH基因。以16SrRNA基因和巨噬细胞GAPDH做为内参基因,将二者的比值作为检测布鲁氏菌进入巨噬细胞数量多少的指标,建立绵羊种布鲁氏菌019株侵染巨噬细胞不同阶段的感染模式。结果表明:在绵羊种布鲁氏菌019株侵染巨噬细胞15min～1h,16SrRNA与GAPDH的比值缓慢上升,侵染1～4h中,16SrRNA与GAPDH的比值急剧上升,说明在此阶段,细菌侵入细胞中的数量显著增多。

To study and research brucella intracellular parasitism mechanism.The test detect the number of intracellular brucellas were described by real-time PCR.Two genes,including B.ovis 019 strain 16S rRNA and mouse RAW264.7 macrophage GAPDH genes,were cloned.Standard curve of two objective genes were described.The infection model of Brucella invading macrophage was built at different stages.The ratio between B.ovis 019 strain 16S rRNA and mouse RAW264.7 macrophage GAPDH genes was detected.The results showed the ratio between 16S rRNA and GAPDH slowly rised during 15 min-1 h after infection.However,the ratio rapidly rised during 1～4 h,which conveyed us the number of intracellular pathogen was significantly enhanced.