高致病性PRRSV GD疫苗株与野毒株双重荧光定量RT-PCR鉴别方法的建立  

Development of a duplex real-time RT-PCR for differential detection of GDr180 vaccine strain from wild-type of highly pathogenic PRRSV

作　　者：张文利[1] 孙丰廷[2] 王海光[1] 刘灿[2] 英聪[2] 宁宜宝[1] 

机构地区：[1]中国兽医药品监察所生药楼检测技术研究室/国家猪瘟参考实验室,北京100081 [2]华中农业大学,重庆武汉430000

出　　处：《中国预防兽医学报》2012年第11期898-902,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家十一五科计支撑计划(2007BAD86B01)

摘　　要：为建立一种能够快速鉴别检测猪繁殖与呼吸综合征病毒(PRRSV)GDr180疫苗株与GD野毒株的双重荧光定量RT-PCR方法,本研究在PPRSV的Nsp2基因和Orf7基因区域分别设计不同荧光标记的MGB探针及特异性引物。这两种MGB探针能够分别特异结合GD野毒株和GDr180疫苗株。通过对PRRSV培养液、感染猪血清和组织样品检测证明了该方法的可行性;采用双重实时荧光定量RT-PCR方法检测PRRSV GDr180疫苗株和GD强毒株时敏感性分别达到19.4拷贝/μL和34.2拷贝/μL;该方法与PRRSV其他8个病毒株和猪瘟病毒、猪伪狂犬病毒、猪圆环病毒2型和猪细小病毒不发生交叉反应,表明该方法具有良好的特异性;因此可以用于PRRSV GDr180疫苗株与野毒株的鉴别检测。To establish a rapid differential detection method between the porcine reproductive and respiratory syndrome virus （PRRSV） GDr180 vaccine strain and its wild type of highly pathogenic PRRSV. Two sets of specific primers and fluorescence dye labeled MGB probes, which were expected to differentiate PRRSV GDr180 vaccine strain from wild type, were designed based on the nucleotide sequence of Nsp2 and ORF7, respectively. The reliability of this method was confirmed by detecting the virus in cell culture, PRRSV infected pig serum and tissue samples. The sensitivity reached to 19.4 copies/μL of GDr180 vaccine virus and 34.2 copies/μL of GD wild virus. Meanwhile, there were no cross-reactions among the 8 different PRRSV strains, classical swine fever virus, pseudo rabies virus, porcine circovirus 2, and porcine parvovirus. In conclusion, the specificity, sensitivity and reliability of this duplex real-time RT-PCR method was feasible for the differential detection of PRRSV GDr180 vaccine strain from its wild type.

关 键 词：高致病性猪繁殖与呼吸综合征活疫苗GDr180 双重荧光RT-PCR MGB探针 鉴别检测 

分 类 号：S852.65[农业科学—基础兽医学]