白斑综合征病毒(WSSV)3种PCR检测方法的灵敏度比较  

Sensitivity comparison of three PCR-based methods for white spot syndrome virus(WSSV) detection

作　　者：肖广侠[1,2] 李战军[1,3] 孟宪红[1] 张庆文[1] 罗坤[1] 孔杰[1] 

机构地区：[1]中国水产科学研究院黄海水产研究所,山东青岛266071 [2]上海海洋大学水产与生命学院,上海200090 [3]大连海洋大学生命科学与技术学院,辽宁大连116023

出　　处：《中国水产科学》2011年第3期667-673,共7页Journal of Fishery Sciences of China

基　　金：国家863计划项目(2010AA10A401);山东省自然科学基金资助项目(ZR2009DQ002);农业部公益性行业科研专项(200803012);中央级公益性科研院所基本科研业务费2009年专项资金项目(2009-chb-04)

摘　　要：为了探讨不同PCR检测方法的灵敏度,分别利用TaqMan实时定量PCR、世界动物卫生组织(OIE)公布的巢式PCR引物(简称OIE)、黄海水产研究所种质资源与工程育种研究室(GB)设计的引物(简称GB)及2种巢式PCR对应的一步法PCR,对具有不同白斑综合征病毒(White Spot Syndrome Virus,WSSV)含量的中国明对虾(Fenneropenaeus chinensis)样品进行检测。结果显示,当使用已知病毒含量的标准品进行检测时,TaqMan实时定量PCR方法可以检测到10个WSSV拷贝;OIE巢式PCR与GB巢式PCR方法分别可检测到104和103个WSSV拷贝;单独使用OIE巢式PCR的外引物和内引物扩增时,分别可检测到5×104和2.5×104个WSSV拷贝;单独使用GB巢式PCR的外引物和内引物进行一步法PCR扩增时,分别可检测到104和5×103个WSSV拷贝。使用上述PCR方法分别对44份未知WSSV含量的样品进行验证,定量PCR方法检测阳性率为84.09%,OIE巢式PCR与GB巢式PCR方法检测的阳性率分别为18.18%和27.27%;单独使用OIE巢式PCR的外引物和内引物扩增检测的阳性率均为15.91%;单独使用GB巢式PCR的外引物和内引物扩增检测的阳性率分别为18.18%和20.45%。根据以上结果,PCR方法检测WSSV的灵敏度由高到低依次为:定量PCR、巢式PCR、一步法PCR。In order to explore the limitations of different PCR methods, we compared the sensitivity of three PCR methods, including TaqMan real-time PCR, Office International Des Epizooties-designed （OIE） and Genetic resource and Breeding Lab of Yellow Sea Fisheries Research Institute-designed nested PCR （GB） and one-step PCR methods, with standard Fenneropenaeus chinensis samples with serial known WSSV load. As a result, TaqMan real-time PCR could detect with certainty 10 WSSV copies per reaction, while 104and 103 copies were needed for OIE and GB nested PCR; 5×104 and 2.5x 104 copies were needed for the outer primer and inner primer amplifica- tion of OIE nested PCR; 104 and 5x103 copies were needed for that of GB nested PCR. Forty-four F. chinensis samples with unknown WSSV load were tested with these PCR methods to validate the sensitivity. The positive ratio of TaqMan real-time PCR was 84.09 %, and those of OIE nested PCR and the GB nested PCR were 18.18% and 27.27%, respectively; the positive ratio of the outer primer and inner primer amplification of OIE nested PCR both were 15.91%; and those of GB nested PCR were 18.18% and 20.45%. According to the results, the sensitivity of PCR methods detecting WSSV from high to low are as follow： TaqMan real-time PCR, nested PCR, one-step PCR. This experiment also provided method-selection strategy and reference data in WSSV diagnose. Key words： PCR testing method; white soot syndrome virus （WSSV）： sensitivitv

关 键 词：PCR检测法 白斑综合征病毒(wssv) 灵敏度 

分 类 号：S945[农业科学—水产养殖]