猪繁殖与呼吸综合征活疫苗中病毒含量荧光定量PCR测定方法的建立及初步应用  

Development and Preliminary Application of Real-time PCR Assay for Detecting Quantitation of PRRSV in Vaccine

作　　者：马志亮[1] 孙亭亭[1] 杨洁[1] 范斌[1] 刘旭川[1] 张以芳[1] 

机构地区：[1]云南农业大学动物科学技术学院,云南昆明650201

出　　处：《中国畜牧兽医》2014年第5期71-76,共6页China Animal Husbandry & Veterinary Medicine

摘　　要：为建立快速、敏感、特异的评价猪繁殖与呼吸综合征（PRRS）活疫苗中病毒含量的方法，根据GenBank中登录的猪繁殖与呼吸综合征病毒（PRRSV）基因序列设计合成了标准品引物和定量引物，RT—PCR扩增PRRSV基因片段并连接到pMD19-T载体上，构建标准品质粒pMD19-TPRRSV。采用SYBRGreenI染料法进行荧光定量PCR检测，分析标准曲线并进行特异性、稳定性、重复性试验。结果显示，该方法检测病毒含量为7．43×10^0～7．43×10^8拷贝／μL，标准品各稀释度质粒拷贝数与Ct值之间相关系数高（R2=0．9989），引物特异性强。应用该方法对6个厂家PRRS活疫苗中的病毒含量进行测定，发现不同厂家生产的疫苗中病毒含量差异较大，最高差异可达47．9倍。本试验建立的检测PRRS活疫苗病毒含量的荧光定量PCR方法可用于疫苗生产过程中及免疫动物前疫苗质量的评价。To establish a rapid, sensitive and specific method for evaluation of porcine reproductive and respiratory syn- drome （PRRS） attenuated vaccine, two pairs of primers were designed and synthesized according to porcine reproductive and respiratory syndrome virus （PRRSV） genes in GenBank. PRRSV genes were obtained by RT-PCR and constructed recombinant plasmids pMD19-T-PRRSV. SYBR Green I method was adopted to improve the Real-time PCR and the method was tested for specificity, repeatability and stability. The results showed that there was a good linear correlation coefficient （R2 =0. 9989） as Ct values ranged from 7.43 ×10^0 to 7.43 ×10^8 copies/μL. Using this method to test PRRS vaccines from different companies, we found that there were significant differences （47.9 times） in the levels of virus among six companies＇ vaccines. The Real-time PCR for detecting PRRS vaccine virus can be used for evaluation of vaccine during production process and animals immunization.

关 键 词：猪繁殖与呼吸综合征活疫苗 荧光定量PCR 应用 

分 类 号：S858.28[农业科学—临床兽医学]