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中国农学通报

中国农学通报 ›› 2013, Vol. 29 ›› Issue (17): 37-41.doi: 10.11924/j.issn.1000-6850.2012-3755

所属专题： 畜牧兽医

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

猪伪狂犬病毒Taqman-MGB荧光定量PCR检测方法的建立及应用

郑敏 毛凝 黄梅清 陈仕龙 陈少莺   

  • 收稿日期:2012-11-19 修回日期:2013-03-12 出版日期:2013-06-15 发布日期:2013-06-15

Establishment and application of TaqMan-MGB fluorescence quantitative PCR for detection of pseudorabies virus

  • Received:2012-11-19 Revised:2013-03-12 Online:2013-06-15 Published:2013-06-15

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摘要： 本研究根据猪伪狂犬病病毒gE基因序列，设计一对特异引物和探针，通过优化反应条件，建立了可区分猪伪狂犬野毒株及基因缺失疫苗株的TaqMan-MGB荧光定量PCR检测方法。结果表明：TaqMan-MGB荧光定量PCR检测方法灵敏度可达2.23×101拷贝/μL，比常规PCR检测方法高100倍；同时检测猪圆环病毒2型(PCV2)、猪瘟病毒(CSFV)、猪繁殖和呼吸综合征病毒(PRRSV)、猪细小病毒（PPV）、均为阴性，无交叉反应；对30份疑似病料的TaqMan荧光定量PCR和普通PCR检测阳性率分别为40%和33%，两者符合率90%。表明该方法灵敏度高、特异性强、重复性好，可同时检测大量样品，可适合于PRV的临床诊断和流行病学调查。

关键词: 序列分析, 序列分析

Abstract: A pairs of primers and a Taqman-MGB probes were designed and synthesized according to the the nucleotide sequence of the gE gene of pseudorabies virus(PRV) available in GenBank，and real-time TaqMan-MGB fluorescence quantitative PCR for distinguishing the wild strain and gene-deleted vaccine strain of PRV was established successfully. It was demonstrated that the established TaqMan-MGB quantitative PCR assay could detect 2.23×101 copys?μL-1 of plasmid DNA . Sensitivity and positive rate for clinical sample of TaqMan fluorescent quantitative PCR were higher than routine PCR,and its sensitivity Was 100 times higher than that of the routine PCR.,and had no cross reaction with classical swine fever virus(CSFV)，porcine reproductive and respiratory syndrome virus(PRRSV) ,porcine cireovirus type 2 (PCV2) and Porcine parvovirus (PPV) The 30 suspected material were detected by TaqMan-MGB fluorescence quantitative PCR and routine PCR , respectively, the positive detection rate were 40% and 33%, the coincidence rate was 90% .The real-time TaqMan fluorescence quantitative PCR assay which is specific，sensitive,rapid and accurate can be used for the diagnosis of PRV infection． Key words：pseudorabies virus(PRV)；TaqMan-MGB；real-time PCR