猪IGF1、EGFR、EGF、β-actin基因荧光定量PCR标准曲线的建立  

Development of the standard curve for detecting pig IGF1,EGFR,EGF,β-actin mRNA with fluorescence quantitative polymerase chain reaction

作　　者：徐环叶 曹玲芝 刘博 陈立功[1] 孟利佳 崔欢 库尔瓦尼古丽.伊明江 董世山[1] 

机构地区：[1]河北农业大学兽医诊断中心/农业部动物疫病病原生物学华北科学观测实验站,河北保定071001 [2]衡水市农牧局,河北衡水053000 [3]伊犁特克斯县畜牧局兽医站,新疆特克斯835500

出　　处：《河北农业大学学报》2017年第5期84-89,共6页Journal of Hebei Agricultural University

基　　金：国家自然基金项目(31470125)

摘　　要：为建立用荧光定量PCR方法检测猪IGF1、EGFR、EGF、β-actin基因表达量的标准曲线,本试验根据目的基因在NCBI上CDS区序列,设计合成引物,构建目的基因重组质粒,采用SYBR GreenI荧光定量RT-PCR的检测方法,构建检测猪IGF1、EGFR、EGF、β-actin基因表达量的标准曲线。结果显示,由pEASY-T1目的基因所构建的标准曲线线性关系良好,线性回归系数(R2)均大于或等于0.99,起始模板与Ct值之间呈良好的线性关系,溶解曲线峰值单一,表明引物具有很好的特异性,结果准确可靠。本研究成功构建了用荧光定量RT-PCR方法检测猪IGF1、EGFR、EGF、β-actin基因表达量的标准曲线,为后续检测基因相对表达量的变化奠定基础。To establish the standard curve for detecting pig IGF1,EGFR,EGF,β-actin expression with fluorescence quantitative polymerase chain reaction,the primers were designed and synthesized according to the pig target gene CDS sequences in NCBI.The recombinant plasmids including target gene were constructed.Then SYBR GreenI fluorescence quantitative RTPCR assay was carried,and the standard curve for detecting pig IGF1,EGFR,EGF,β-actinexpression was constructed.The results indicated that standard curve made by pEASY-T1-target gene had an excellent linear relationship（R2≥0.99）between logarithmic concentrations of the plasmid DNA and corresponding Ct values,indicating that the PCR amplification efficiency washigh within range.The single melting temperature peak corresponded to a specific product,revealing that the results were accurate and reliable.Our results show that the standard curve for detecting pig IGF1,EGFR,EGF,β-actin expression with fluorescence quantitative RTPCR was developed successfully,which laid a foundation to detect the gene relative expression changes.

关 键 词：猪 修复基因 RT-PCR 标准曲线 重组质粒 

分 类 号：Q781[生物学—分子生物学]