摘要
菊花是世界最重要的园艺作物之一,兼具有茶用、药用、食用及观赏等多种经济价值。我国是栽培菊花的起源地,也是主要种植生产区。病毒病害是菊花生产过程中最主要病害,目前已知的菊花病毒及类病毒有20多种,对菊花产业危害巨大。建立快速、高效的检测方法是病害防控的必要条件。前期调查显示番茄斑萎病毒(Tomato spotted wilt orthotospovirus,TSWV)、番茄不孕病毒(Tomato aspermy virus,TAV)、菊花B病毒(Chrysanthemum virus B,CVB)、菊花矮化类病毒(Chrysanthemum stunt viroid,CSVd)和菊花褪绿斑驳类病毒(Chrysanthemum chlorotic mottle viroid,CChMVd)是北京地区菊花中主要病毒及类病毒种类。自NCBI数据库下载以上病毒/类病毒的基因序列,利用不同病毒保守区域,设计开发出特异性引物,建立了5种病毒/类病毒同时检测的多重RT⁃PCR体系;并对反应体系中引物对的含量配比、退火温度、模板含量等因子进行了优化。试验结果显示,TSWV、TAV、CVB、CChMVd、CSVd的引物对浓度分别为300 nmol/L、50 nmol/L、200 nmol/L、700 nmol/L、600 nmol/L,模板用量0.5 ng/μL,退火温度55.3℃时,可以同时扩增出片段大小为分别485 bp、376 bp、292 bp、220 bp、110 bp的目的条带,特异性良好,结果判读简单清晰。进一步的比较分析显示,其与单一病毒检测的RT⁃PCR体系的检测灵敏性相当。综上,本研究构建的多重RT⁃PCR体系可以高效、快速、灵敏的检测菊花中常见的5种病毒及类病毒,并且检测成本大幅降低,为病毒的快速、低成本检测提供了方法。
Chrysanthemum(Chrysanthemum×morifolium)is one of the most important horticultural crops in the world.It has many economic uses,including use in tea,medicine,food and ornamental.Cultivated Chrysanthemums were originated in China,which is an important producing area for chrysanthemum.Diseases caused by virus and viroid are the most important disease in chrysanthemum production,and at least 20 viruses and viroid have been reported to infect chrysanthemum.Detection is a prerequisite and foundation for virus and viriods.Preliminary research indicated that tomato spotted wilt virus(TSWV),tomato aspermy virus(TAV),chrysanthemum virus B(CVB),chrysanthemum stunt viroid(CSVd)and chrysanthemum chlorotic mottle viroid(CChMVd)are main types of viruses and viroids in Chrysanthemum in Beijing.The gene sequences for above viruses and viroids were downloaded from NCBI database and special PCR primers were designed according to their conserved regions.A multiplex RT⁃PCR system was developed for simultaneous detection of the five viruses and viroids amplified fragment of TSWV、TAV、CVB、CChMVd and CSVd with fragment size of 485bp、376bp、292bp、220 bp and 110 bp respectively.System optimization indicated that the optimal primer concentrations was 300 nmol/L、50 nmol/L、200 nmol/L、700 nmol/L and 600 nmol/L,respectively.The best template dosage was 0.5 ng/μL and the most suitable annealing temperature was 55.3℃.The detection sensitivity of the multiplex RT⁃PCR system developed in this study was equivalent to that of PCR for single virus detection.In conclusion,we established a multiplex RT⁃PCR system for five common virus/viroid simultaneous determination and provided a rapid and low⁃cost virus/viroids detection method.
作者
张利英
王森
陈东亮
喻锌
姜宝龙
程钰涵
李彦慧
黄丛林
ZHANG Liying;WANG Sen;CHEN Dongliang;YU Xin;JIANG Baolong;CHENG Yuhan;LI Yanhui;HUANG Conglin(Institute of Grassland,Flowers and Ecology,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097,China;Beijing Functional Flower Engineering Technology Research Center,Beijing 100097,China;College of Landscape and Tourism,Hebei Agriculture University,Baoding 071000,China;Beijing Genesand Biotech Co.,Ltd,Beijing 102209,China;College of Landscape Architecture,Beijing University of Agriculture,Beijing 102206,China)
出处
《病毒学报》
CAS
CSCD
北大核心
2023年第4期1045-1052,共8页
Chinese Journal of Virology
基金
现代农业产业技术体系北京市创新团队项目(项目号:BAIC09⁃2023,题目:北京市景观休闲农业创新团队。
关键词
菊花
病毒
类病毒
多重RT⁃PCR
Chrysanthemum×morifolium
Virus
Viroid
Multiplex RT⁃PCR
Detection
