柑橘3种病毒类病原多重RT-PCR检测技术的建立及应用  

A Multiplex RT-PCR Assay for Detecting Three Pathogens Infecting Citrus Plants

作　　者：袁琳凯 马崇欢 李丁山 陈志炜 江宵烽 丁新伦[1] 张洁[1] 吴祖建[1] YUAN Linkai;MA Chonghuan;LI Dingshan;CHEN Zhiwei;JIANG Xiaofeng;DING Xinlun;ZHANG Jie;WU Zujian(Institute of Plant Virology,Fujian Agriculture and Forestry University/State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops,Fuzhou,Fujian 350002,China)

机构地区：[1]福建农林大学植物病毒研究所/闽台作物有害生物生态防控国家重点实验室,福建福州350002

出　　处：《福建农业学报》2024年第3期339-344,共6页Fujian Journal of Agricultural Sciences

基　　金：福建省三明市横向项目(KH210264A、KH210295A、KH220062A)。

摘　　要：【目的】建立柑橘黄化脉明病毒(citrus yellow vein clearing virus,CYVCV)、柑橘衰退病毒(citrus tristeza virus,CTV)和啤酒花矮化类病毒(hop stunt viroid,HSVd)的多重RT-PCR检测体系。【方法】设计多重RT-PCR引物,分析其特异性,确定其最佳浓度比、最适退火温度及灵敏度,在此基础上对福建地区的柑橘样品进行检测。【结果】确定了CYVCV-F/R、CTV-F/R和HSVd-F/R等3对引物的最佳浓度比例为1∶1∶2,最适退火温度为52.9℃,灵敏度结果显示该体系可检测模板稀释到10^(-2)的阳性样品。应用该体系对采自福建部分地区的157份柑橘样品进行检测,结果发现,CYVCV、CTV和HSVd的检出率分别为47.1%、56.7%和22.9%。【结论】成功建立了柑橘CYVCV、CTV和HSVd病原的多重RT-PCR检测方法,为该类病害的检测提供准确、快速的检测方法。【Objective】A multiplex RT-PCR assay to simultaneously detect citrus yellow vein clearing virus(CYVCV),citrus tristeza virus(CTV),and hop stunt viroid(HSVd)was developed.【Method】Multiplex RT-PCR primers were designed,and their specificity was analyzed.The optimal concentration ratio,annealing temperature and sensitivity of primers were determined.Moreover,citrus samples from Fujian region were detected by the established multiplex RT-PCR.【Result】The optimized concentration ratio of CYVCV-F/R,CTV-F/R,and HSVd-F/R primers was 1:1:2,and the annealing temperature at 52.9℃for the assay.The assay displayed a sensitivity at 10^(-2)dilution and the positive detections on CYVCV of 47.1%,on CTV of 56.7%,and on HSVd of 22.9%on 157 detected samples.【Conclusion】A rapid,accurate,applicable multiplex RT-PCR method for CYVCV,CTV,and HSVd detections was successfully established.

关 键 词：柑橘黄化脉明病毒 柑橘衰退病毒 啤酒花矮化类病毒 多重RT-PCR 

分 类 号：S432.1[农业科学—植物病理学]