中国兽医科学2017，Vol.47Issue(10)：1292-1298,7.DOI:10.16656/j.issn.1673-4696.2017.10.015
山羊IFN-γ和TNF-α基因SYBR Green Ⅰ实时荧光定量RT-PCR检测方法的建立及应用
Establishment and application of SYBR Green Ⅰ real-time fluorescence quantification RT-PCR for IFN-γ and TNF-α genes of goat
摘要
Abstract
Interferon-γ(IFN-γ) and tumor necrosis factor-α(TNF-α) of goat were selected according to GenBank to design specific primers to amplify the target genes.The two kinds of genes were cloned into pMD-19-T vector to obtain the respective positive clone plasmids,which were used as standards to create a standard curve.Melting curve analysis as well as sensitivity,specificity and reproducibility experiments were conducted.With the proposed method,the gene transcription levels of IFN-γ and TNF-α at different time points were detected through the established method,after concanavalin A (ConA) stimulated peripheral blood mononuclear cells(PBMC) in healthy goat.The results showed that,there was a good linear relationship between the Ct value and the concentration of the amplification curve when the dilution of plasmid was 1 × 109 copies/μ L～1 × 105 copies/μ L,and the correlation coefficient was≥-0.996.The melting curve analysis showed that the product was specific and reproducible.The mRNA transcription peak of IFN-γ occurred at 2 h and 48 h,and IFN-γ transcription was 1.26× 105 copies/μL at 2 h and 8.56× 104 copies/μL at 48 h.The mRNA transcription of TNF-α showed a decline trend in 0 h～24 h,and the peak occurred at 48 h.The transcription amount was 2.73×105 copies/μL.The results would provide a technical platform for the quantitative analysis of IFN-γand TNF-α.关键词
山羊/细胞因子/实时荧光定量RT-PCR/SYBR Green ⅠKey words
caprine/cytokines/real-time fluorescence quantitative PCR/SYBR Green Ⅰ分类
农业科技引用本文复制引用
李婷,张益,鲜思美,包细明,冯将,李鹏飞..山羊IFN-γ和TNF-α基因SYBR Green Ⅰ实时荧光定量RT-PCR检测方法的建立及应用[J].中国兽医科学,2017,47(10):1292-1298,7.基金项目
贵州省科技计划课题[黔科合LH字(2014)7667] （2014）
贵州大学2015年大学生创新创业训练计划项目[贵大(省)创字2015(005)] （省）
贵州省动物疫病防控与兽医公共卫生保障科技创新人才团队项目[黔科合人才团队(2015)4016号] （2015）
