高致病性猪繁殖与呼吸综合征病毒实时荧光定量RT-PCR鉴别诊断方法的建立  

Real-time RT-PCR assay for detection and differentiation of classical and mutational PRRS virus of Northern American type

作　　者：孔留五[1,2] 张桂红[1] 姜增固[2] 康艳梅[1] 秦宏阳[1] 曹宗喜[1] 张得玉[1] 张翔峰[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]广州市良种猪场,广东广州510540

出　　处：《广东畜牧兽医科技》2009年第6期25-29,共5页Guangdong Journal of Animal and Veterinary Science

基　　金：广东省自然科学基金(5006678);广东省科技计划项目(2007A0203006)

摘　　要：根据GenBank收录的PRRSV基因序列,利用PRRSV经典变异株与缺失变异株NSP2基因序列特点,设计合成两对特异性引物,经RT-PCR扩增后,用T4连接酶将目的片段与pMD18-T载体连接,并转化到大肠杆菌DH-5a株感受态细胞。提取的质粒经PCR、酶切和测序鉴定,证实为PRRSV缺失变异株与经典变异株的阳性重组质粒。将重组标准质粒进行10倍系列稀释后作为模板,通过实时荧光定量PCR法(Real-timePCR),建立了PRRSV缺失变异株与经典变异株的标准曲线及其直线回归方程,并确定其最佳读板温度。该方法具有线性关系好、特异性强、敏感性高、重复性好等特点,为鉴别诊断及分析PRRSV缺失变异株与经典变异株感染猪体内病毒的绝对含量提供了技术手段。Two pairs of primers were designed and synthesized according to the nsp2 genes classical and mutational PRRS virus available in GenBank. A real-time quantitative reverse transcriptase PCR （QT-PCR） assay was established for the rapid detection, differentiation and quantitation of PRRS virus. The PCR product of the target gene was ligated with pMD18-T vector and transformed to competent cell E. Coli DH5α . The purified plasmid was identified to be combinant positive plasmid of classical and mutational PRRS virus after restriction enzyme digestion and sequencing. The standard curve was established by detecting recombinant plasmids with different concentration. The results showed that the real time RT-PCR assay was specific, sensitive and repeatable. It provided one effective method for differentiating classical and mutational PRRS virus and detecting the virus load in pigs.

关 键 词：PRRSV 实时荧光定量 鉴别诊断 绝对含量 

分 类 号：S814[农业科学—畜牧学]