荧光实时定量RT-PCR观察伤寒杆菌鞭毛z66和d/j抗原基因表达方法的建立

目的：构建荧光实时定量RT-PCR测定伤寒杆菌鞭毛抗原z66和d／j编码基因mRNA的方法。方法：根据伤寒杆菌鞭毛抗原z66和d／i编码基因序列，设计引物扩增其读码框架内片段，克隆进pGEM-TEasy质粒。质粒经PCR鉴定后，纯化并定量，作为荧光实时定量PCR的标准品，并制作标准曲线。利用不同渗透压下的鞭毛素基因表达差异，比较三种随机引物(6、8、10核苷酸)与特异性引物的逆转录效果，以确定一种最佳的随机引物用于多基因表达观察。结果：标准曲线有较好的线性(r=0．999)，cDNA和标准品的PCR产物溶解曲线峰值一致。用8核苷酸随机引物进行逆转录的敏感度高于6、10核苷酸随机引物，低于特异性引物，但用其观测z66抗原基因和d／j抗原基因在高、低渗时表达变化趋势与用特异性引物测得结果一致。结论：成功构建用8核苷酸随机引物进行逆转录同时测定伤寒杆菌鞭毛抗原z66和d／j基因表达的荧光实时定量RT-PCR方法。

A Method of Fluoresce Real time RT-PCR for Investigating the z66 and d or j Antigen Genes Expression of Salmonella Enterica Serovar Typhi

Objective: For investigating the flagellin gene expression of Salmonella enterica serovar Typhi, a method of fluoresce real time RT-PCR was set up to detect mRNA levels of z66 and d or j antigen genes. Methods: According the genomic information of Salmonella, specific primers of z66 and d or j antigen genes were designed. Two amplicons from z66 antigen positive strain of S. enterica serovar Typhi were cloned into the pGEM-T easy vector as the standard template DNA of flagellin genes. The standard curve for detecting mRNA levels of z66 and d or j antigen genes was studied, and the three random primers (6,8 and 10-mer)and the specific primer were investigated by comparing the detection results in Salmonella incubated in different osmolarity conditions. Results: The correlative coefficient of the standard of the method was 0. 999, and the peak of melting curve of the amplicon from cDNA was identical to that from standard template DNA. The detection sensitivity of the method using 8-mer random primer was highest among three random primers, and it was lower than that using the specific primer. There was no significant difference between detection methods using 8-mer random primer and specific primers in investigation results of flagellin gene expression variations of S. enterica serovar Typhi, which were incubated in different osmolarities. Conclusion:The real time RT-PCR method using 8-mer random primer was much better for investigating mRNA levels of flagellin genes of S. enterica serovar Typhi.