鸭源PKC基因荧光定量PCR检测方法的建立
Establishment of Real-time Fluorescence Quantitative PCR Method for Detecting PKC Gene in Duck

为建立基于鸭源宿主细胞的蛋白激酶PKC基因的实时荧光定量PCR检测方法。根据GenBank数据库的PKC基因设计、合成特异性引物，通过PCR技术扩增目的基因片段，构建重组质粒GV pXT19-T-PKC，并以其为阳性标准品建立荧光定量PCR检测方法和标准曲线，进行重复性、特异性和敏感性验证。结果显示，荧光定量PCR标准曲线为y=－3.334 0x+44.199，相关系数为0.995 4，扩增效率为99.19%；熔解曲线仅出现单特异峰，未检测到H5亚型/H7亚型/H9亚型禽流感病毒疫苗毒株、新城疫病毒和鸭肝炎病毒等参考毒株的核酸样本的荧光信号。说明，建立的荧光定量PCR方法具有良好的稳定性、特异性和灵敏性。
To develop a SYBR GreenⅠ Real-Time PCR method assay for detecting the PKC gene in duck, the specific primers were designed and synthesized accorded to the PKC gene in the GenBank. and the target gene fragment obtained by PCR. Then the recombinant plasmid GV pXT19-T-PKC were constructed. The real-time fluorescent quantitative PCR method and the standard curve were established, then analyzed its reproducibility, specificity and sensitivity. The linear relationship of standard curve was y=-3.334 0x+44.199, and the correlation coefficient was 0.995 4, the amplification efficiency was 99.19%. There were a single specific peak appeared in the melting curve, and the fluorescence signal were not detected in the H5, H7, and H9 subtype of avian influenza virus, Newcastle disease virus and duck hepatitis virus.All the results indicated the Real-Time fluorescent quantitative PCR method for PKC gene in ducks is excellent stability, specificity and sensitivity.