猪流行性腹泻病毒TaqMan荧光定量RT—PCR检测方法的建立  

Development of TaqMan Fluorescence Quantitative RT—PCR Assay for Detection of Porcine Epidemic Diarrhea Virus

作　　者：张志[1] 刘自立[2] 董雅琴[1] 于美芳[1] 刘爽[1] 吴发兴[1] 邵卫星[1] 李晓成[1] 王树双[1] 

机构地区：[1]中国动物卫生与流行病学中心,山东青岛266032 [2]云南农业大学,云南昆明650201

出　　处：《中国动物检疫》2014年第4期76-79,84,共5页China Animal Health Inspection

基　　金：科技基础性工作专项(2012FY111000);青岛市民生计划项目(13-1-3-91-nsh)

摘　　要：本为快速特异地检测猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV),根据猪流行性腹泻病毒N基因序列设计合成引物和探针,通过对荧光定量RT-PCR反应条件的优化,建立了TaqMan荧光定量RT—PCR检测PEDV的方法。用阳性质粒10倍梯度稀释的标准品绘制标准曲线,结果显示,该方法的标准曲线的相关系数为0.99,检测敏感性达到26.1拷贝/μL,且具有很好的特异性和重复性,对猪瘟病毒、猪传染性胃肠炎病毒、猪繁殖与呼吸综合征病毒等检测结果均为阴性,批内和批间变异系数均小于2.5%。对43份现地病料的检测结果也表明该方法(检出28份)比常规RT—PCR方法(检出12份)敏感性更高,为该病的快速诊断和流行病学调查提供了有效手段。A TaqMan fluorescence quantitative RT--PCR assay was developed by designing and synthesizing a pair of primers and a labelled probe according to the conservative sequences of N gene of porcine epidemic diarrhea virus. The coefficient of standard curve was 0.99 testified by a 10 diluted series of recombinant plasmid. The sensitivity of detection limit was upto 26.1 copies/u L without cross reaction with classical swine fever virus （CSFV） , porcine transmissible gastroenteritis virus （TGEV） and porcine reproductive and respiratory syndrome virus （PRRSV） .The variation coefficient ofintra-batch and inter-batch were both below 2.5%, indicating good reproducibility. This assay were further tested with 43 field samples resulting in 28 positive ones, 12 more than those by the routine RT--PCR. The method could be used in PEDV infection diagnosis and investigation.

关 键 词：猪流行性腹泻病毒 TAQMAN探针 荧光定量RT--PCR 

分 类 号：S858.28[农业科学—临床兽医学]