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| 白斑综合征病毒(WSSV)3种PCR检测方法的灵敏度比较 | |
| 引用本文： | 肖广侠,李战军,孟宪红,张庆文,罗坤,孔杰.白斑综合征病毒(WSSV)3种PCR检测方法的灵敏度比较[J].中国水产科学,2011,18(3):667-673. |
| 作者姓名： | 肖广侠 李战军 孟宪红 张庆文 罗坤 孔杰 |
| 作者单位： | 1. 中国水产科学研究院黄海水产研究所,山东青岛266071;上海海洋大学水产与生命学院,上海200090 2. 中国水产科学研究院黄海水产研究所,山东青岛266071;大连海洋大学生命科学与技术学院,辽宁大连116023 3. 中国水产科学研究院黄海水产研究所,山东青岛,266071 |
| 基金项目： | 国家863计划项目，山东省自然科学基金资助项目，农业部公益性行业科研专项，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 |
| 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l0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. |
| 关 键 词： | PCR检测法 白斑综合征病毒(WSSV) 灵敏度 |
| 修稿时间： | 2011/5/9 0:00:00 |
Sensitivity comparison of three PCR-based methods for white spot syndrome virus (WSSV) detection |
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| XIAO Guangxi,LI Zhanjun,MENG Xianhong,ZHANG Qingwen,LUO Kun,KONG Jie.Sensitivity comparison of three PCR-based methods for white spot syndrome virus (WSSV) detection[J].Journal of Fishery Sciences of China,2011,18(3):667-673. | |
| Authors: | XIAO Guangxi LI Zhanjun MENG Xianhong ZHANG Qingwen LUO Kun KONG Jie |
| Affiliation: | 1. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;2. College of Fisheries and Life Technology, Shanghai Ocean University, Shanghai 200090, China;3. College of Life Science and Technology, Dalian Ocean U |
| Abstract: | 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 Yellow Sea Fisheries Research Institute-designed nested PCR (GB) and one-step PCR methods, with standard samples with serial known WSSV load. As a result, 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 2.5×104 copies were needed for the outer primer and inner primer amplification of OIE nested PCR; 104 and 5×103 copies were needed for that of GB nested PCR. Forty-four samples with unknown WSSV load were tested with these PCR methods to validate the . 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. |
| Keywords: | PCR testing method white spot syndrome virus (WSSV) sensitivity |
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