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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 
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 NameAffiliationE-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
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