| 苜蓿花叶病毒RT-PCR和RT-qPCR检测技术体系的建立与应用 |
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| 引用本文：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.植物保护学报,2022,49(2):515-527 |
| DOI：10.13802/j.cnki.zwbhxb.2022.2021040 |
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| 作者 | 单位 | E-mail | | 高艳玲 | 东北农业大学农学院, 哈尔滨 150030
黑龙江省农业科学院经济作物研究所, 哈尔滨 150086 | | | 范国权 | 黑龙江省农业科学院经济作物研究所, 哈尔滨 150086 | | | 程胜群 | 东北农业大学农学院, 哈尔滨 150030 | | | 张威 | 黑龙江省农业科学院经济作物研究所, 哈尔滨 150086 | | | 邱彩玲 | 黑龙江省农业科学院经济作物研究所, 哈尔滨 150086 | | | 申宇 | 黑龙江省农业科学院经济作物研究所, 哈尔滨 150086 | | | 聂先舟 | 加拿大农业和农业食品部弗雷德里克顿研究发展中心, 新不伦瑞克 弗雷德里克顿 E3B4Z7 | | | 白艳菊 | 黑龙江省农业科学院经济作物研究所, 哈尔滨 150086 | yanjubai@163.com | | 吕文河 | 东北农业大学农学院, 哈尔滨 150030 | luwenhe60@163.com |
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| 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 |
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| 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 |
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| 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 |
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