| 猪流感病毒一步法荧光定量RT-PCR诊断方法的建立 |
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| 引用本文： | 徐敏,李志强,黄元,陈晶,陈少勤,张健騑,赵亚华,宣华,向华.猪流感病毒一步法荧光定量RT-PCR诊断方法的建立[J].中国兽医科学,2011(10). |
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| 作者姓名： | 徐敏 李志强 黄元 陈晶 陈少勤 张健騑 赵亚华 宣华 向华 |
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| 作者单位： | 广东省农业科学院兽医研究所广东省兽医公共卫生公共实验室;华南农业大学生命科学学院; |
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| 基金项目： | 广东省农业科学院院长基金项目(20090203); 广东省科技厅项目(2009B020600001、2009A020101006、2006B20801002、2006A20301006、2006B21101004); 东莞市科技局项目(200910810130) |
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| 摘 要： | 为了建立一种在猪流感流行病学调查和临床诊断中应用的快速高效的诊断方法,根据猪流感病毒(swine influenza virus,SIV)M基因的保守序列,设计并合成1对特异性引物和1条特异性TaqMan探针,建立了以RNA为反应模板的一步法荧光定量RT-PCR诊断方法。使用含有M基因的重组质粒作为标准品绘制标准曲线,该曲线效率值为2.014,误差值为0.005。试验结果表明,该方法灵敏度极高,最低可检测到10 copies的核酸;临床样品检测结果也显示其灵敏度高于普通PCR和病毒分离法;特异性强,对猪群常见的其他6种病毒检测结果均为阴性。临床样品检测显示,该方法操作简单,可以用RNA作为模板直接检测,节省时间,而且特异性好、灵敏度高,是可以应用于猪流感流行病学调查的一种可靠的诊断方法。
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| 关 键 词： | 猪流感病毒 一步法荧光定量RT-PCR TaqMan探针 |
Development of one-step real-time quantitative RT-PCR approach for the detection of swine influenza virus |
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| XU Min,LI Zhi-qiang,HUANG Yuan,CHEN Jing,CHEN Shao-qin,ZHANG Jian-fei,ZHAO Ya-hua,XUAN Hua,XIANG Hua.Development of one-step real-time quantitative RT-PCR approach for the detection of swine influenza virus[J].Veterinary Science in China,2011(10). |
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| Authors: | XU Min LI Zhi-qiang HUANG Yuan CHEN Jing CHEN Shao-qin ZHANG Jian-fei ZHAO Ya-hua XUAN Hua XIANG Hua |
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| Institution: | XU Min1,LI Zhi-qiang2,HUANG Yuan1,CHEN Jing1,CHEN Shao-qin1,ZHANG Jian-fei1,ZHAO Ya-hua2,XUAN Hua1,XIANG Hua1(1.Veterinary Public Health Laboratory of Guangdong,Veterinary Medicine Institute,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China,2.College of Life Science,South China Agricultural University,Guangzhou 510642,China) |
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| Abstract: | In order to develop a rapid and high-performance assay method for epidemiological study and clinical diagnosis of swine influenza virus,a one-step real-time quantitative RT-PCR method based on a TaqMan probe was developed.Conserved regions in the M gene of swine influenza viruses served as targets for primers and TaqMan probe.Concentrations of primers and probe were optimized to improve the sensitivity and specificity of reactions.A series of dilutions of recombinant plasmid were prepared and used to genera... |
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| Keywords: | swine influenza virus one-step real-time quantitative RT-PCR TaqMan probe |
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