中国兽医科学2013，Vol.43Issue(2)：184-189,6.
三种鸡细胞因子mRNA实时荧光定量RT-PCR检测方法的建立与应用
Establishment and application of real time fluorescent quantitative RT-PCR method for the detection of three chicken cytokines
摘要
Abstract
In order to establish the assay of SYBR Green I real-time fluorescent quantitative RT-PCR for detecting three important chicken cytokines ChIL-12,ChIL-15 and ChIL-18, four pairs of specific primers were designed according to the sequences of ChIL-12,ChIL-15,ChIL-18 and reference gene ChGAPDH from GenBank to amplify the objective gene. The four genes were respectively cloned to the pMD18-T vector. The corresponding plasmids were identified by sequencing, and they were then used as quantitative template to construct the standard curve and to analyze the melting curve, detection sensitivity, specificity and repeatability. The results showed that C, value of the four gene had good linear relationship (R2 > 0. 985) with the dilution ranging from 1 × 102 to 1 × 1010 copies/μL. The melting curve displayed a single peak and good repeatability. The established assay was used to detect the transcriptional level of ChIL-12 and ChIL-18 mRNA in the spleen of chickens immunized by nucleic acid vaccine pcDNA-Ea3-lE. The results showed that mRNA level of ChIL-12 and ChIL-18 in immunized group were both significantly higher than that in control group(P<0. 01). The research provides the platform for quantitative analysis of chicken cytokine ChIL-12,ChIL-15 and ChIL-18.关键词
鸡/细胞因子/SYBR Green Ⅰ/实时荧光定量 RT-PCRKey words
chicken/cytokine/SYBR Green Ⅰ /real time fluorescent quantitative RT-PCR分类
农业科技引用本文复制引用
高铭扬,马春丽,马德星,李广兴,张瑞莉..三种鸡细胞因子mRNA实时荧光定量RT-PCR检测方法的建立与应用[J].中国兽医科学,2013,43(2):184-189,6.基金项目
国家自然科学基金资助项目(30901061) （30901061）
中国博士后科学基金项目(2011M501007) （2011M501007）
东北农业大学博士启动基金项目(2010RCB38) （2010RCB38）
哈尔滨市青年科技创新基金项目(2012RFQXN003) （2012RFQXN003）
