实时定量RT-PCR检测马动脉炎病毒  

Detection of Equine Arteritis Virus by Real-time Reverse Transcription-PCR Assay

作　　者：梁成珠[1,2] 杨松[3,4] 高宏伟 朱来华[1,2] 蔡梅红[3] 王谦滨 陈溥言[3] 

机构地区：[1]青岛出入境检验检疫局 [2]南京农业大学动物医学院,江苏南京210095 [3]南京农业大学动物医学院 [4]沈阳农业大学畜牧兽医学院,辽宁沈阳110161

出　　处：《中国兽医学报》2005年第2期141-144,共4页Chinese Journal of Veterinary Science

基　　金：国家质检总局科研项目(2002IK009-03)

摘　　要：选取马动脉炎病毒 (EAV )基因组中高度保守的开放阅读框 7(ORF7)序列 ,设计了特异性引物和 Taq Man探针 ,利用一步法的实时定量 RT- PCR来定量检测 EAV。使用含有选定检测序列的克隆标准品做标准曲线 ,证明该方法可以检测出含有 5 (4.77)个 RNA分子 ,即可从组织培养液 (TCF)中检出一般含有 5 PFU/μL 病毒拷贝。对猪生殖呼吸道综合征病毒、马疱疹病毒无交叉反应 ,特异性好。用疫苗用种毒 (Bucyrus标准株 )肌肉注射于马驹后 ,定量 RT- PCR比病毒分离试验更能灵敏地检出呼吸道排出的病毒。对 4 5 6份临床鼻腔分泌物样品检测 ,定量 RT- PCR检出 6份阳性 ,而病毒分离只检出 2份阳性。一步法实时定量 RT- PCR检测样品中的 EAV,在快捷、准确、方便和高通量方面优于细胞培养病毒分离的检测方法 ,可以作为检测马病毒性动脉炎 (EVA )A one-step fluorogenic RT-PCR(5′-nuclease probe-based) assay using a primer/TaqMan probe set designed against a highly conserved region of open reading frame 7(ORF7) of EAV genome was developed for the specific detection of EAV.The assay had an analytical sensitivity of 5 molecules of EAV RNA allowing the detection of EAV in clinical samples or tissue culture fluid(TCF) containing about 5 PFU/μL.Following intramuscular injection of two ponies with vaccine virus Bucyrus strain,the excretion of virus in respiratory passages can be detected more sensitively by the flunorogenic RT-PCR than by virus isolation test.The one-step RT-PCR method specifically detected EAV in samples from Customs and quarantine departments with rapider,more accurate,more convenient and higher sample throughout than our routine diagnostic procedures of ELISA and virus isolation in cell culture.Therefore it may be considered as a valuable tool to complement the routine diagnostic procedures for EAV diagnosis.

关 键 词：马 动脉炎病毒 实时RT-PCR TAQMAN探针 定量检测 病毒性动脉炎 

分 类 号：S858.21[农业科学—临床兽医学] S852.659.6[农业科学—兽医学]