PRRSV经典与高致病性毒株RT-PCR鉴别检测方法的建立  

Development of a RT-PCR for detection and differentiation of classical and high-pathogenic PRRSVs based on Nsp2 gene

作　　者：肖跃强[1] 管宇[1,2] 唐娜[1] 魏凤[1] 杨慧 曲光刚[1] 沈志强[1,2] 

机构地区：[1]山东省滨州畜牧兽医研究院,山东滨州256600 [2]山东绿都生物科技有限公司,山东滨州256600

出　　处：《中国兽医学报》2010年第7期873-877,共5页Chinese Journal of Veterinary Science

基　　金：山东省自然科学基金资助项目(Y2006D26)

摘　　要：根据GenBank登录的经典与高致病性PRRSVNsp2序列,设计了1对引物,所扩增的序列包含高致病性毒株缺失部分,用于区分经典与高致病性毒株,并对扩增条件进行了优化。结果表明,应用该方法,能从经典与高致病性PRRSV基因组中分别扩增出573,483bp的特异性片段,病毒基因组混合后能够同时扩增;并可以检测出1.8TCID50/100μL经典毒株及0.6TCID50/100μL高致病毒株的病毒RNA;对CSFV,JPV,PEDV,TGEV,RV,PPV,PRV,PCV-2等常见猪病毒病病原的鉴别诊断均为阴性;对分离的8株PRRSV进行检测,3株为经典毒株,5株为高致病毒株;对采集的59份确诊病料进行了检测,21份为经典毒株感染,38份为高致病毒株感染。本研究建立的RT-PCR诊断方法,具有特异、灵敏、快速等特点,可用于区分PRRSV经典与高致病毒株。A reverse transcription-polymerase chain reaction （RT-PCR） was developed for detection and differentiation of classical and high-pathogenic American porcine reproductive and respiratory syndrome viruses （PRRSV）.A pair of primers corresponding to the highly conserved regions of Nsp2 gene of PRRSV genome were synthesized,the amplified fragement containing the deleted sequences.According to the length of the amplified fragements,the classical and high-pathogenic PRRSV could be differentiated.And the amplication conditions were optimized.A fragment of 573 or 483 bp was amplified from genomic RNA of classical or high-pathogenic PRRSV respectively,and these two fragments were also simultaneously amplified from the mixed RNA samples.No specific products were achieved after differentiating detections of porcine originated viral pathogens including CSFV,JEV,PEDV,TGEV,RV,PPV,PRV and PCV2.The RT-PCR could detect 1.8 TCID50 classical or 0.6 TCID50 high-pathogenic virus RNA.Eight isolates were differentiated with this method,electrophoresis and sequencing results showed that three strains were divided into the classical group while the others belong to high-pathogenic group.Forthermore,twenty-one of fifty-nine field positive samples were detected to be classical virus infenction,and the others to be high-pathogenic virus infection.The RT-PCR method can be used for differentiating classical and high-pathogenic PRRSVs,and provides great value for clinical diagnosis.

关 键 词：PRRSV NSP2基因 RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]