苜蓿花叶病毒RT-PCR和RT-qPCR检测技术体系的建立与应用  

Development and application of RT-PCR and RT-qPCR for detection of alfalfa mosaic virus

作　　者：高艳玲[1,2] 范国权[2] 程胜群 张威[2] 邱彩玲[2] 申宇[2] 聂先舟 白艳菊[2] 吕文河[1] Gao Yanling;Fan Guoquan;Cheng Shengqun;Zhang Wei;Qiu Cailing;Shen Yu;Nie Xianzhou;Bai Yanju;LüWenhe(College of Agriculture,Northeast Agricultural University,Harbin 150030,Heilongjiang Province,China;Industrial Crops Institute,Heilongjiang Academy of Agricultural Sciences,Harbin 150086,Heilongjiang Province,China;Fredericton Research and Development Centre,Agriculture and Agri-Food Canada,Fredericton E3B4Z7,New Brunswick,Canada)

机构地区：[1]东北农业大学农学院,哈尔滨150030 [2]黑龙江省农业科学院经济作物研究所,哈尔滨150086 [3]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

出　　处：《植物保护学报》2022年第2期515-527,共13页Journal of Plant Protection

基　　金：财政部和农业农村部国家现代农业产业技术体系(CARS-09-P16);黑龙江省农业科学院院级课题(2018YYYF022);黑龙江省农业科学院农业科技创新跨越工程(HNK2019CX19-06)。

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1.45×10^(1) copies/μL和1.46×10^(1) 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1.15×10^(2) 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3.04×10^(6)~1.67×10^(8) 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2.94×10^(2)~4.78×10^(8) 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8.64×10^(4)~4.76×10^(7)、2.13×10^(3)~1.50×10^(5)和8.08×10^(3)~4.96×10^(5) 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4.71×10^(3)~3.44×10^(4)、2.22×10^(2)~1.32×10^(5)和5.41×10^(1)~8.08×10^(2) 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。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 1.45×10^(1) and 1.46×10^(1) 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 1.15×10^(2) 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 3.04×10^(6)-1.67×10^(8) 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 2.94×10^(2)-4.78×10^(8) copies/μL,and no clear distribution pattern was found.The concentrations of AMV RNA 1,RNA 2,and RNA 3 in thrips were 8.64×10^(4)-4.76×10^(7),2.13×10^(3)-1.50×10^(5),and 8.08×10^(3)-4.96×10^(5) 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 were4.71×10^(3)-3.44×10^(4),2.22×10^(2)-1.32×10^(5),and 5.41×10^(1)-8.08×10^(2) 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.

关 键 词：苜蓿花叶病毒 TaqMan探针RT-qPCR EvaGreen RT-qPCR RT-PCR 马铃薯 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]