鲑传染性贫血病毒TaqMan实时荧光定量RT-PCR检测方法的建立及应用  

Establishment and application of a TaqMan real time RT-PCR for the detection of infectious salmon anaemia virus

作　　者：肖璐[1] 杨苗[2] 林华[2] 陈世界[2] 安微[2] 王印[1] 刘荭[3] 严玉宝[2] 胡娟[2] 王军[2] 邬旭龙[1] 李丹丹[4] XIAO Lu YANG Miao LIN Hua CHEN Shi-jie AN Wei WANG Yin LIU Hong YAN Yu-bao HU Juan WANG Jun WU Xu-long LI Dan-dan(Sichuan Entry-Exit Inspection and Quarantine Bureau, Chengdu 610041, China College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen 518045 ,China Hainan Entry-Exit Inspection and Quarantine Bureau ,Haikou 570311, China)

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]四川出入境检验检疫局,四川成都610041 [3]深圳出入境检验检疫局,广东深圳518045 [4]海南出入境检验检疫局,海南海口570311

出　　处：《中国兽医科学》2017年第7期915-919,共5页Chinese Veterinary Science

基　　金：四川出入境检验检疫局科技项目(SK201503)

摘　　要：为建立快速、定量的鲑传染性贫血病毒(ISAV)检测方法,基于ISAV片段7 ORF1编码的结构蛋白(NS)保守基因序列,设计特异性引物和TaqMan探针,通过反应条件优化,建立ISAV的TaqMan荧光定量RT-PCR检测方法,对其特异性、灵敏度及重复性进行评估,将其应用于口岸进境大西洋鲑报检样本ISAV检测中,并与OIE推荐的检测方法的检测结果进行比较。结果显示,本方法特异性强,灵敏度高,最低检测灵敏度达1.31×10~2copies/L。重复性试验结果显示,本方法的批内变异系数小于1%。299份2014—2016年间进口大西洋鲑报检样本的检测结果显示,ISAV的总检出率为6.35%(19/199),该结果与OIE推荐的检测方法的结果完全一致,且利用本方法测定的19份阳性样本的Ct值普遍低于OIE推荐检测方法测得的Ct值。结果表明,本研究中所建立的方法高效、敏感、特异、重复性好,在口岸检疫中具有广阔的应用前景,对于ISAV的鉴别诊断具有一定的参考意义。To establish a method for the rapid and quantitative detection of infectious salmon anaemia virus（ISAV）,one pair of primers and TaqMan probe were designed based on NS,which is conservative and coded by segment seven ORF1.After optimizing reaction conditions,a TaqMan real time PCR for detection of ISAV was established,then the sensitivity,specificity and repeatability were estimated,and the method was used in salmon specimens which were collected between 2014 to 2016 from Chinese ports.The detection results were compared with that of the method recommended by OIE.The result showed the TaqMan method had high specificity and repeatability,and the minimum detection limit reached to 1.31×10^2copies/L.The coefficient of variation was less than 1%,and 299 clinical samples detection results showed the positive detection rate of ISAV was 6.35%（19/299）,which was consistent with the results byOIE,and the Ctof the method was lower than that of the method of OIE in the detection of the 19 positive specimens.This method is efficient,sensitive,specific and stable,which has broad application prospects in quarantine department, and it is meaning for the identification of ISAV.

关 键 词：鲑传染性贫血病毒 TAQMAN 荧光定量RT-PCR 口岸检疫 

分 类 号：S852.659.5[农业科学—基础兽医学]