猪繁殖与呼吸综合征病毒TaqMan荧光定量RT-PCR方法的建立与应用  

Development and application of TaqMan one-step fluorescence quantitative RT-PCR assay for detection of porcine reproductive and respiratory syndrome virus

作　　者：刘长龙[1] 张建武[1] 韦祖樟[1] 袁世山[1] 

机构地区：[1]中国农业科学院上海兽医研究所猪呼吸系统疾病研究室,上海200241

出　　处：《中国预防兽医学报》2010年第11期858-861,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：十一五支撑项目(2006BAD06A01)

摘　　要：为快速检测猪繁殖与呼吸综合征病毒(PRRSV),本研究针对PRRSV的N蛋白基因保守序列,设计特异性引物和TaqMan探针,以体外转录的RNA作为标准品,设计并优化了检测PRRSV的TaqMan荧光定量PCR方法。应用该方法检测其它主要猪病病原,结果均呈阴性;针对PRRSV阳性RNA的检测灵敏度可以达到10拷贝,比常规PCR灵敏10倍～100倍;结果表明,本实验建立的PRRSV TaqMan荧光定量PCR方法具有良好的特异性、敏感性和重复性。应用建立的方法测定攻毒猪血清中病毒的含量,免疫攻毒组和非免疫攻毒组在第7 d血清中病毒的载量差异不显著,而在第14 d差异显著,免疫攻毒组猪血清中病毒载量明显低于未免疫组。A quantitative TaqMan one-step RT-PCR assay for detecting Porcine reproductive and respiratory syndrome virus（PRRSV） was developed using two pairs of primers based on N protein nucleotide sequence of PRRSV.This method was highly specific and did not amplify non-specific products from other swine pathogens.It could detect 10 copies of RNA at least,and was 10 to 100 fold higher sensitive than conventional RT-PCR.The method was used to determine the virus load in pig serum and showed that the immunized pigs had significantly lower virus load than control animals on the 14 days after virus challenge.

关 键 词：猪繁殖与呼吸综合征病毒 荧光定量PCR TAQMAN探针 RNA标准品 

分 类 号：S852.65[农业科学—基础兽医学]