猪繁殖与呼吸综合征病毒和猪圆环病毒2型基因重组质粒实时荧光定量PCR标准曲线的构建  

Construction of recombinant porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 gene recombinant plasmids and establishment of standard curves for detection of PRRSV/PCV2 genes using Real-time PCR

作　　者：施开创[1] 郭鑫[1] 杨汉春[1] 李焕荣[1] 盖新娜[1] 陈艳红[1] 查振林[1] 

机构地区：[1]中国农业大学农业部预防兽医学重点开放实验室,北京海淀100094

出　　处：《中国兽医杂志》2007年第4期10-12,共3页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金项目(30371072)

摘　　要：根据猪繁殖与呼吸综合征病毒(PRRSV)ORF7和猪圆环病毒2型(PCV2)ORF1的核苷酸序列设计特异性引物,经RT-PCR/PCR扩增,分别扩增出大小为308 bp和417 bp的部分基因片段。用T4连接酶将目的片段与pGEM-Teasy载体系统连接,并转化到大肠杆菌DH-5α株感受态细胞。提取的质粒经PCR、酶切和测序鉴定,证实为PRRSV和PCV2的阳性重组质粒。将重组标准质粒10倍系列稀释后作为模板,通过实时荧光定量PCR方法(Real-time PCR),建立了PRRSV、PCV2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,为分析PRRSV和PCV2共感染猪体内病毒的绝对含量提供了必要的技术平台。Here we report the construction of porcine reproductive and respiratory syndrome virus （PRRSV） and establishment of porcine circovirus type 2 （PCV2） gene recombinant plasmids and standard curves for detection of PRRSV/PCV2 genes using real-time fluorescent quantitative PCR （Real-time PCR）. A 308-bp fragment of PRRSV ORF7 gene and a 417-bp fragment of PCV2 ORF1 gene were amplified by RT-PCR/PCR from viral cell cultures using specific primers. The fragments were ligated with pGEM-T easy vector and then transformed to E coll. DH-Sa. Both PRRSV and PCV2 recombinant plasmids were acquired successfully, based on the results of amplification by PCR, digestion with restriction endonuclease EcoR I and gene sequencing. Their standard curves and the corresponding linear regression equations were obtained by Real-time PCR assay. In addition, the optimal plate-reading temperatures were also determined. The results indicated that the standard curves were shown to be of high linearity, specificity, sensitivity and reproducibility, and they provided powerful tools for quantification of PRRSV and PCV2 loads in pigs after co-infection with PRRSV and PCV2.

关 键 词：PRRSV PCV2 实时荧光定量PCR 重组质粒 标准曲线 

分 类 号：S858.285.3[农业科学—临床兽医学]