苜蓿花叶病毒RT-PCR和RT-qPCR检测技术体系的建立与应用 Click here to download the full text Citation：高艳玲,范国权,程胜群,张威,邱彩玲,申宇,聂先舟,白艳菊,吕文河.苜蓿花叶病毒RT-PCR和RT-qPCR检测技术体系的建立与应用.Journal of Plant Protection,2022,49(2):515-527 Hits: Download times: Author Name Affiliation E-mail GAO Yan-ling  College of Agriculture, Northeast Agricultural University, Harbin 150030, Heilongjiang Province, China Industrial Crops Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang Province, China    FAN Guo-quan  Industrial Crops Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang Province, China    CHENG Sheng-qun  College of Agriculture, Northeast Agricultural University, Harbin 150030, Heilongjiang Province, China    ZHANG Wei  Industrial Crops Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang Province, China    QIU Cai-ling  Industrial Crops Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang Province, China    SHEN Yu  Industrial Crops Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang Province, China    NIE Xian-zhou  Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, Fredericton E3B4Z7, New Brunswick, Canada    BAI Yan-ju  Industrial Crops Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang Province, China  yanjubai@163.com  LU: Wen-he  College of Agriculture, Northeast Agricultural University, Harbin 150030, Heilongjiang Province, China  luwenhe60@163.com  中文摘要:为准确检测侵染马铃薯核心种苗和种薯的苜蓿花叶病毒(alfalfa mosaic virus,AMV),以AMV马铃薯分离株为阳性材料,建立AMV RNA 1、RNA 2和RNA 3的反转录-聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)和基于EvaGreen及TaqMan探针的反转录实时荧光定量PCR (reverse transcription real-time fluorescent quantitative PCR,RT-qPCR)检测技术,比较3种方法的检测灵敏度,并测定不同马铃薯品种6种组织和桃蚜Myzus persicae、蓟马体内AMV RNA 1、RNA 2和RNA 3的含量。结果表明:建立的TaqMan探针RT-qPCR法对RNA 1和RNA 2的检测灵敏度分别为1.45×101 copies/μL和1.46×101 copies/μL,分别是EvaGreen RT-qPCR法和RT-PCR法的10倍;建立的TaqMan探针RT-qPCR法对RNA 3的检测灵敏度与EvaGreen RT-qPCR法相同,为1.15×102 copies/μL,是RT-PCR法的10倍。丽薯15号6种组织中AMV RNA 1、RNA 2和RNA 3的含量为3.04×106~1.67×108 copies/μL,以叶片中病毒平均含量最高;中薯21号6种组织中AMV RNA 1、RNA 2和RNA 3的含量为2.94×102~4.78×108 copies/μL,未发现明显分布规律。蓟马体内AMV RNA 1、RNA 2和RNA 3的含量分别为8.64×104~4.76×107、2.13×103~1.50×105和8.08×103~4.96×105 copies/μL,RNA 1的含量高于RNA 2和RNA 3。桃蚜体内AMV RNA 1、RNA 2和RNA 3的含量分别为4.71×103~3.44×104、2.22×102~1.32×105和5.41×101~8.08×102 copies/μL,RNA 3的含量最低。表明RT-PCR法、TaqMan探针RT-qPCR法和EvaGreen RT-qPCR法均可有效扩增马铃薯6种组织和2种昆虫中的AMV。 中文关键词:苜蓿花叶病毒  TaqMan探针RT-qPCR  EvaGreenRT-qPCR  RT-PCR  马铃薯   Development and application of RT-PCR and RT-qPCR for detection of alfalfa mosaic virus Author Name Affiliation E-mail GAO Yan-ling  College of Agriculture, Northeast Agricultural University, Harbin 150030, Heilongjiang Province, China Industrial Crops Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang Province, China    FAN Guo-quan  Industrial Crops Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang Province, China    CHENG Sheng-qun  College of Agriculture, Northeast Agricultural University, Harbin 150030, Heilongjiang Province, China    ZHANG Wei  Industrial Crops Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang Province, China    QIU Cai-ling  Industrial Crops Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang Province, China    SHEN Yu  Industrial Crops Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang Province, China    NIE Xian-zhou  Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, Fredericton E3B4Z7, New Brunswick, Canada    BAI Yan-ju  Industrial Crops Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang Province, China  yanjubai@163.com  LU: Wen-he  College of Agriculture, Northeast Agricultural University, Harbin 150030, Heilongjiang Province, China  luwenhe60@163.com  Abstract:In order to detect alfalfa mosaic virus(AMV) infecting plantlets in vitro and seed potatoes accurately, the reverse transcription-polymerase chain reaction(RT-PCR), EvaGreen and TaqMan probe reverse transcription real-time fluorescent quantitative PCR(RT-qPCR) were developed to detect AMV RNA 1, RNA 2, and RNA 3. The sensitivities of three methods were compared, and the contents of AMV RNA 1, RNA 2, and RNA 3 in six potato tissues of different potato varieties, aphids, and thrips were determined. The sensitivities of TaqMan probe RT-qPCR for RNA 1 and RNA 2 were 1.45×101 and 1.46×101 copies/μL recombinant plasmids, respectively, which were ten times those of EvaGreen RT-qPCR and RT-PCR methods. The sensitivity of TaqMan probe RT-qPCR for RNA 3 was the same as that of EvaGreen RT-qPCR, which was 1.15×102 copies/μL, ten times that of RT-PCR. The concentrations of RNA 1, RNA 2, and RNA 3 in six tissues of Lishu 15 were 3.04×106-1.67×108 copies/μL, with the average virus content highest in the leaves; but in Zhongshu 21, the contents of AMV RNA 1, RNA 2, and RNA 3 were 2.94×102-4.78×108 copies/μL, and no clear distribution pattern was found. The concentrations of AMV RNA 1, RNA 2, and RNA 3 in thrips were 8.64×104-4.76×107, 2.13×103-1.50×105, and 8.08×103-4.96×105 copies/μL, respectively, with the content of RNA 1 being relatively higher than that of RNA 2 and RNA 3. The concentrations of AMV RNA 1, RNA 2, and RNA 3 in Myzus persicae were4.71×103-3.44×104, 2.22×102-1.32×105, and 5.41×101-8.08×102 copies/μL, respectively, with RNA 3content lowest. These results indicated that AMV in six potato tissues and two insect species could be amplified effectively by RT-PCR, TaqMan probe and EvaGreen RT-qPCR. keywords:alfalfa mosaic virus  TaqMan probe RT-qPCR  EvaGreen RT-qPCR  RT-PCR  potato View Full Text  View/Add Comment  Download reader

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