实时荧光定量PCR检测鳜IgM mRNA标准品质粒的构建

Construction of the Standards for Detecting IgM mRNA of Siniperca chuatsi with Real-time Quantitative PCR

利用SYBR Green I荧光定量PCR技术,建立鳜IgM-DNA定量标准品的制备方法。从浸泡免疫后的鳜头肾中提取总RNA逆转录合成cDNA,对目的片段进行PCR扩增、电泳纯化、T-A克隆及测序鉴定。将所得预期质粒梯度稀释后构建标准曲线并进行融解曲线分析。结果表明,当标准品的浓度在3.29×102～3.29×108拷贝/μL时,模板浓度与循环阈值(Ct)间的线性关系良好,相关系数r2达到0.999;融解曲线分析显示具特异的单个峰,表明扩增产物特异性非常好。此法制备的重组质粒标准品可用于对鳜IgM基因的转录水平进行测定。

A method to monitor the expression of IgM mRNA using real-time quantitative PCR was established. Total RNA was extracted from the head kidney of Siniperca chuatsi immunized with immersion then cDNA was synthesized by reverse transcription. Target sequence was amplified by PCR and cloned into T vector. The recombinant plasmids were used as template to make standard curve. The results showed a good correlation coefficient （r^2= 0.999） between template concentration and Ct value. The melting curve appeared a single peak, indicating accurate amplification in the quantitative PCR. It is viable that the plasmid standards can be used to detect change of IgM transcription.