实时定量PCR快速检测对虾白斑综合症病毒  

DETECTION AND QUANTIFICATION OF WHITE SPOT SYNDROME VIRUS IN SHRIMPS USING REAL-TIMET PCR ASSAY

作　　者：毕道荣[1] 岳志芹[1] 梁成珠[2] 凌宗帅[2] 赵巍[2] 张健[2] 邓明俊[2] 刘荭[3] 徐彪[2] 郑小龙[2] 何俊强[3] 

机构地区：[1]中国动物卫生与流行病学中心,山东青岛266114 [2]山东出入境检验检疫局检验检疫技术中心,山东青岛266002 [3]深圳出入境检验检疫局水生动物疫病国家重点实验室,广东深圳518010

出　　处：《山东农业大学学报（自然科学版）》2009年第1期54-58,共5页Journal of Shandong Agricultural University：Natural Science Edition

基　　金：国家质量监督检验疫总局资助项目(2006IK003);山东出入境检验检疫局资助项目(SK200686)。

摘　　要：根据对虾白斑综合症病毒(White Spot Syndrome Virus,WSSV)的保守序列,利用Primer Express2.0软件设计引物和Taqman探针,建立了WSSV的实时定量PCR检测体系。构建含有WSSV目的扩增片段的质粒为标准品,进行定量PCR扩增,结果表明标准品浓度在1.5×10~7～1.5×10~1个拷贝之间有"S"型扩增曲线,检测限为15个拷贝。标准曲线中模板浓度(X)与Ct值的关系为:Ct=-3.77 lgX+43.73,相关系数R^2=0.9967。该检测方法特异性强,对传染性皮下和造血器官坏死病毒(IHHNV)、斑节对虾杆状病毒(MBV)、肝胰腺细小病毒(HPV)以及对虾基因组DNA没有扩增反应。运用该方法对100尾对虾样品进行检测,14个对虾样品为阳性,而运用巢式PCR检测只有4个对虾样品为阳性。实时定量PCR方法检测WSSV,快速、特异、灵敏,在虾病的临床检测上具有重要意义。A real - time quantitative PCR assay was developed for detection and quantification of White Spot Syndrome Virus （WSSV）. Primers and probe were designed based on the conserved region of WSSV with Primer Express 2.0 software. The real - time PCR assay had a detection limit of 15 DNA copies, with a dynamic range of detection between 1.5 10^7 - 1.5 10^1 copies. The linear relationship between virus concentration （X） and Ct was Ct = - 3.77 lgX ＋ 43.73 with the correlation coefficient R^2 = 0. 9967. The primers and probe were specific for WSSV and did not react with either Infectious Hypodermal and Hematopoietic Necrosis Virus （ IHHNV ） , Monodon Baculovirus （MBV） , Hepatopancreatic Parvovirus （HPV） , or shrimp genomie DNA. The real -time PCR assay detected 14 positive WSSV samples from 100 shrimps,.while the nested PCR detected 4 positive samples. The real -time PCR assay is highly sensitive, specific, time -saving and easy to handle. It is considered to be a powerful tool for the rapid detection and quantification of WSSV in shrimps.

关 键 词：实时定量PCR 白斑综合症病毒 对虾 

分 类 号：S945.4[农业科学—水产养殖]