小反刍兽疫疫苗病毒含量荧光定量PCR检测方法的建立及初步应用

Establishment and preliminary application of fluorescence quantitative PCR for detection of PPRV in vaccine

为了建立快速、敏感、特异的小反刍兽疫(peste des petits ruminants,PPR)活疫苗中病毒含量的检测方法,本研究根据GenBank数据库中登录的小反刍兽疫病毒(peste des petits ruminants virus,PPRV)基因序列设计合成特异性引物,采用RT-PCR扩增PPRV基因片段,并克隆至pUC57载体上,构建标准品质粒p UC57-N-PPRV,再采用SYBR GreenⅠ染料法进行实时荧光定量PCR(real-time PCR)检测,绘制标准曲线并进行特异性、稳定性和重复性试验,同时应用于检测4批不同厂家生产的PPR活疫苗病毒含量。结果显示,用该方法检测病毒含量为2.61×10^(1)~2.61×10^(9)copies/μL的标准品,各稀释度质粒拷贝数与C_(t)值之间呈良好的线性关系,且相关系数高(R^(2)=0.999),说明引物特异性强。4批不同厂家生产的PPR活疫苗的病毒含量水平差异显著(P<0.05)。结果表明,该方法具有耗时短、灵敏度高、特异性强且稳定等优点,可用于检测PPR活疫苗中病毒含量水平。

The purpose of the study was to establish a rapid,sensitive and specific method for detecting virus content of the live peste des petits ruminants(PPR)vaccine.In this study,specific primers were designed and synthesized according to peste des petits ruminants virus(PPRV)genes in the GenBank database.PPRV genes were obtained by RT-PCR and constructed recombinant plasmids pUC57-N-PPRV.SYBR GreenⅠmethod was used for the real-time PCR detection,the standard curve was drawn and the specificity,stability and reproducibility were verified.It is also used to detect the virus content of 4 batches of the live PPR vaccines.The results showed that there was a linear relationship between the number of plasmid copies and the C_(t) value in the range of 2.61×10^(1)—2.61×10^(9) copies/μL,and the correlation coefficient was high(R^(2)=0.999),it indicated that the primers were highly specific.The level of virus content of four batches of the live PPR vaccines produced by different manufacturers was significantly different(P<0.05).This shows that this method has the advantages of short-time,high sensitivity,strong specificity and stability,and can be used to detect the virus content in the live PPR vaccine.