3种瘤胃纤维分解菌实时定量PCR方法的建立

为探索以减毒胞内侵袭菌介导的黏膜免疫对宿主动物胃肠道微生态的影响，本试验以白色瘤胃球菌、黄化瘤胃球菌和产琥珀酸丝状杆菌3种主要瘤胃纤维分解菌16S rRNA分别设计引物，以山羊瘤胃液提取细菌总DNA，分别扩增3种纤维分解菌目的DNA片段，并连接至pMD-18 T Vector上，经PCR和测序鉴定后，以不同稀释度的重组质粒为模板进行荧光定量PCR反应。结果显示，扩增得到的3种瘤胃纤维分解菌目的片段与已知菌种相应片段的同源性大于99%；以不同稀释度重组pMD-18 T为模板建立的荧光定量PCR扩增曲线差异明显，绘制标准曲线的相关系数均接近1，熔解曲线均呈单一峰值。因此，本试验成功建立了3种瘤胃主要纤维分解菌的实时定量PCR方法，为减毒胞内侵袭菌介导的黏膜免疫研究奠定了基础。瘤胃纤维分解菌；Real-time PCR；标准曲线；

Development of a Real-time PCR Approach for Quantification of Three Cellulolytic Bacteria in Rumen

Study on the impact of intestinal microbial ecology of host animals by mucosal immunity with attenuated invasived bacteria，based on the 16S rRNA gene sequences of three cellulolytic bacteria including Ruminococcus albus,Ruminococcus flavefaciens and Fibrobacter succinogene,the primers were designed.Ruminal microbial total DNA was isolated from rumen content，and amplified by PCR.The PCR products was linked with pMD-18T Vector to construct recombinant plasmid and transfected into Escherichia coli.Target plasmids were selected by blue-white selection and their specificity was identified by direct PCR and DNA sequencing.The serial gradient concentrations of plasmid DNA were used to Real-time PCR.Results showed that neucleotide homology of 16S rRNA of three cellulolytic bacteria were higher than 99%.There were obvious differences in amplification curves of Real time PCR by the gradient concentrations of the recombinant plasmid，the correlation coefficient of the three standard curves were close to 1.The melting curves of Real-time PCR displayed a single peak.It was indicated that the standard plasmids and curves were constructed successfully and can be applied to the future research of mucosal immunity with attenuated Salmonella bacteria.