猪流行性腹泻病毒分离鉴定与实时荧光定量RT-PCR检测方法的建立  

Isolation,Identification and Development of Real-time RT-PCR for Detecting Porcine Epidemic Diarrhea Virus

作　　者：唐金明[1,2] 陈书琨[1] 林庆燕[1] 黄新亚[3] 卢体康[1] 孙洁[1] 阮周曦[1] 曾少灵[1] 祁振强 吕建强[1] 杨俊兴[1] 曹琛福[1] 张彩虹[1] 刘建利[1] 廖立珊[1] 花群义[1] 秦智锋[1] 

机构地区：[1]深圳出入境检验检疫局,广东深圳518045 [2]华南农业大学,广东广州510642 [3]湖南农业大学,湖南长沙410000 [4]深圳市宝舜泰生物医药股份有限公司,广东深圳市518001

出　　处：《动物医学进展》2014年第8期31-35,共5页Progress In Veterinary Medicine

基　　金：深圳市技术创新计划资助项目(CXZZ20130320154210829)

摘　　要：采用Vero-76细胞系从临床样品小肠内容物中分离到1株猪流行性腹泻病毒(PEDV),通过RT-PCR进行了鉴定。针对PEDV,参照保守的M基因序列设计了特异性引物和探针,建立了针对PEDV特异性的实时荧光定量RT-PCR检测方法(PEDV-rRT-PCR)。所建立的方法特异性强,与传染性胃肠炎病毒、轮状病毒、猪细小病毒等猪主要病原体无交叉反应;方法灵敏度高,检测灵敏度达到重组质粒10-8稀释度(约20cop);重复性好,组内变异系数≤0.26%。20份田间样品检测结果表明,所建立的PEDV-rRTPCR方法快速、灵敏,检测结果准确可靠,适合用于PEDV的快速检测和流行病学调查。A porcine epidemic diarrhea virus（PEDV）strain was isolated with Vero-76 cell line from small intestine contents of swine with clinical signs and identified by RT-PCR assay.Primers and probe were designed according to the conserved M gene of PEDV,and a Taqman real-time RT-PCR（rRT-PCR）assay was developed.The sensitivity and specificity of PEDV-rRT-PCR assay were evaluated.The specificity of rRT-PCR assays developed had no cross reaction with transmissible gastroenteritis virus,rotavirus group A,porcine parvovirus and others.The sensitivity of PEDV-rRT-PCR was 10^-8 dilution of recombinant plasmid with about 20 copies.The assay has a good repeatability with coefficient of variation within group less than or equal to 0.26%.Twenty field samples tested with the assay indicated that PEDV-rRT-PCR was specific,sensitive and rapid.The developed assay is an excellent method for routine detection of PEDV and could be used to monitor and survey PED.

关 键 词：猪流行性腹泻病毒 病毒分离 实时荧光定量RT-PCR 

分 类 号：S852.28[农业科学—基础兽医学] S852.659.6[农业科学—兽医学]