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

中国畜牧兽医

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

马志亮，孙亭亭，杨洁，范斌，刘旭川，张以芳

（云南农业大学动物科学技术学院，云南昆明 650201）

收稿日期:2013-11-12 出版日期:2014-05-20 发布日期:2014-06-25
通讯作者: 张以芳，教授，研究方向：微生物学与免疫学。E-mail: zyfkm@qq.com
作者简介:马志亮（1983—），男，山东人，硕士生，研究方向：微生物学与免疫学。

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

MA Zhi-liang, SUN Ting-ting, YANG Jie, FAN Bin, LIU Xu-chuan, ZHANG Yi-fang

(College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China)

Received:2013-11-12 Online:2014-05-20 Published:2014-06-25

摘要/Abstract

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-T-PRRSV。采用SYBR GreenⅠ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7.43×10⁰～7.43×10⁸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。

关键词:

猪繁殖与呼吸综合征活疫苗; 荧光定量PCR; 应用

Abstract: To establish a rapid, sensitive and specific method for evaluation of porcine reproductive and respiratory syndrome (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 GreenⅠ method was adopted to improve the Real-time PCR and the method was tested for specificity, repeatability and stability. The results shwed that there was a good linear correlation coefficient (R²=0.9989) as Ct values ranged from 7.43×10⁰ to 7.43×10⁸ 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.

Key words:

porcine reproductive and respiratory syndrome attenuated vaccine; Realmso-bidi-font-family: 宋体" lang="EN-US">-time PCR; application

马志亮，孙亭亭，杨洁，范斌，刘旭川，张以芳. 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[J]. 中国畜牧兽医.

MA Zhi-liang, SUN Ting-ting, YANG Jie, FAN Bin, LIU Xu-chuan, ZHANG Yi-fang. Development and Preliminary Application of Real-time PCR Assay for Detecting Quantitation of PRRSV in Vaccine[J]. .

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