中文摘要：

用质粒制备绝对定量PCR标准曲线存在着作为标准品的双链环状质粒分子与待测样品单链线性cDNA分子结构不一致的问题。为了消除二者之间的差异,确保绝对定量PCR测定方法的准确性,我们在用质粒制备标准曲线和测定未知样品中特定基因mRNA分子拷贝数时进行了两个改进：1）用酶切的方法使环状质粒线性化,用线性质粒制备标准曲线;2）将未知样品cDNA扩增出一条正义链,再与作为标准曲线的质粒标准品同时开始荧光定量PCR循环。实验结果表明,1）具有相同拷贝数的环状和线性质粒在一起进行定量PCR测定时,环状质粒所得的Ct值大于线性质粒所得的Ct值（P〈0.05）;2）本研究测定的cDNA样品中先扩增一次测出的起始拷贝数均大于未先扩增一次的测定结果。样品起始拷贝数两次测定结果的差异分析表明5个样品中有3个样品的测定差异达到了显著水平（P〈0.05）。应用该方法改进的鸡GATA5基因绝对定量PCR的标准曲线能够准确地反应目的产物扩增。经改进的质粒制备绝对定量PCR标准曲线的方法更为简便易行,并提高了测定的准确性。

英文摘要：

Using plasmid vector to build standard curve for absolute quantification PCR（AQ-PCR） might induce error because of the molecular difference between super-helix double-strand plasmid DNA and linear single-strand cDNA sample.To increase the exactitude of the AQ-PCR,we improved the method by adding two steps during the quantity assay procession of target gene mRNA copy in unknown samples.1） The plasmid was digested to be linear by restriction enzymes for building standard curve;2） The unknown cDNA samples were amplified to make a sense strand,followed the fluorescence quantitative PCR cycle with standard plasmid together.The results indicated that 1） The same copy of circular and linear plasmid started AQ-PCR test at the same time,the Ct value of circular plasmid was larger than that of linear plasmid（P0.05）;2） The measured starting copy numbers of cDNA samples amplified once were larger than that of cDNA samples unamplified.The measured results of starting copy number between amplified and unamplified showed that there have 3 of 5 samples were significant differences（P0.05）.Using these improvements,the standard curve of chicken GATA5 gene was improved and reflected accurately the amplification of purpose products.Our improved method of using plasmid to build standard curve for AQ-PCR was more feasible,and the measure was more accuracy.