白斑综合征病毒(WSSV)3种PCR检测方法的灵敏度比较

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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
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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;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
作者简介:肖广侠(1984−), 男, 硕士研究生, 主要从事水产动物遗传育种工作. E-mail: gxxiao@163.com
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基金项目: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).

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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

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摘要:

检测方法的灵敏度实时定量(引物、黄海水产研究所种质资源与工程育种研究室简称对具有不同白斑综合征病毒, TaqMan10巢式单独使用的外引物和内引物扩增时单独使用的外引物和内引物进行一步法分别可检测到3PCRWSSVPCR单独使用的外引物和内引物扩增检测的阳性率均为单独使用的外引物和内引物扩增检测的阳性率分别为。根据以上结果的灵敏度由高到低依次为、巢式。

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.

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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
XIAO Guangxia, LI Zhanjun, MENG Xianhong, ZHANG Qingwen, LUO Kun, KONG Jie.[J]. Journal of Fishery Sciences of China,2011,18(3):667-673

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在线发布日期: 2011-05-09
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