表达新城疫病毒F基因的重组鸡痘病毒实时荧光定量PCR检测方法的建立

Development of real-time PCR method for detection of recombinant fowlpox virus coexpressing F gene of Newcastle disease virus

采用荧光染料SYBR Green渗入法,通过对real-time PCR反应条件进行优化,建立了real-timePCR检测方法,用该方法定量分析新城疫病毒(NDV)F基因在重组鸡痘病毒(rFPV-F-VP0)第1、5、10、15、20代次中的表达整合情况。结果表明,建立的标准曲线呈现良好的重复性和特异性,与模板浓度呈现良好的线性关系。在线性浓度范围内,随着模板量的减少,其对应的Ct值相应增大,0.99<相关系数(r2)<1,0.8<扩增效率(E)<1.2,熔解曲线为单一特征峰型。本试验精确定量了外源基因在重组鸡痘病毒中的表达水平,为阐明重组鸡痘病毒的表达机理提供了理论基础。

In order to quantitatively analyze the expression of F gene of Newcastle disease virus（NDV） in recombinant fowlpox virus rFPV-F-VP0,the method with good reproducibility,high specificity and sensitivity standard was established after the real-time PCR reaction conditions were optimized by fluorescent dying methods of SYBR Green.The expression level of NDV F gene in the recombinant fowlpox virus rFPV-F-VP0 was quantitatively analyzed in passages 1,5,10,15 and 20,respectively.The results demonstrated that the standard curve established with the recombinant plasmid showed a fine linear relationship between threshold cycle and template concentration with good reproducibility and specificity.In the linear range of dilution,the dissociation curve was specific and the corresponding Ct value increased accordingly with the reduction in the amount of the template.The correlation coefficient and the amplification efficiency was 0.99 r21 and 0.8 E1.2,respectively.This technique accurately quantified the level of exogenous gene expression in the recombinant fowlpox virus,and provided a theoretical basis to clarify the expression mechanism of the recombinant fowlpox virus.