大蒜病毒多重RT-PCR检测体系的建立与应用

doi:10.12190/j.issn.2096-1197.2023.01.03

北方农业学报 ›› 2023, Vol. 51 ›› Issue (1): 22-30.doi: 10.12190/j.issn.2096-1197.2023.01.03

大蒜病毒多重RT-PCR检测体系的建立与应用

赵永强, 樊继德, 陆信娟, 刘灿玉, 张碧薇, 杨青青, 葛洁, 史新敏, 杨峰

江苏徐淮地区徐州农业科学研究所,江苏徐州 221121

收稿日期:2022-09-20 出版日期:2023-02-20 发布日期:2023-05-22
通讯作者: 杨峰（1975—）,男,研究员,博士,主要从事大蒜育种及栽培技术的研究工作。
作者简介:赵永强（1986—）,男,副研究员,硕士,主要从事植物病理学方面的研究工作。
基金资助:
财政部和农业农村部：国家现代农业产业技术体系资助; 江苏省苏北科技专项（XZ-SZ202160）; 江苏现代农业（蔬菜）产业技术体系（JATS〔2021〕048）

Development and application of a multiplex RT-PCR detection system for garlic viruses

ZHAO Yongqiang, FAN Jide, LU Xinjuan, LIU Canyu, ZHANG Biwei, YANG Qingqing, GE Jie, SHI Xinmin, YANG Feng

Xuzhou Institute of Agricultural Sciences in Jiangsu Xuhuai Area,Xuzhou 221121,China

Received:2022-09-20 Online:2023-02-20 Published:2023-05-22

摘要/Abstract

摘要： 【目的】建立一套多重RT-PCR方法,用于同时检测和鉴别大蒜大田植株和组培苗5种病毒/病毒组。【方法】根据相关文献和GenBank 公布的参考病毒株序列,设计了5对分别针对青葱潜隐病毒（Shallot latent virus,ShLV）、大蒜普通潜隐病毒（Garlic common latent virus,GarCLV）、韭葱黄条病毒（Leek yellow stripe virus,LYSV）、洋葱黄矮病毒（Onion yellow dwarf virus,OYDV）和青葱X病毒属病毒（allexiviruses）的特异性引物,扩增产物大小分别为592、431、338、265、190 bp。利用建立的多重RT-PCR方法,分别对24份大蒜组培苗和48份大田样本进行病毒检测分析。【结果】通过单重/多重PCR及序列比对验证了检测系统的特异性。通过退火温度和引物比例优化,确定退火温度为56.6 ℃且ShLV、GarCLV、LYSV、OYDV、allexiviruses引物体积比为10∶8∶3∶3∶16时,扩增效果最好。5种病毒/病毒组的最低检测浓度均为1.0×10² copies/μL。大蒜组培苗病毒检测结果具有丰富的多样性,大田样品被至少2种病毒/病毒组感染,病毒/病毒组之间可以清晰区分。【结论】开发的多重RT-PCR方法可以快速、有效地检测和鉴别大蒜ShLV、GarCLV、LYSV、OYDV和allexiviruses这5种病毒/病毒组的感染情况。

关键词: 大蒜, 病毒, 多重RT-PCR, 检测体系

Abstract: 【Objective】To establish a multiplex RT-PCR system for simultaneous detection and identification of 5 viruses/virus groups in garlic field plants and tissue cultured plantlets.【Methods】Based on relevant literature and the reference virus sequences registered in GenBank,5 sets of specific primers were designed for Shallot latent virus（ShLV）,Garlic common latent virus（GarCLV）,Leek yellow stripe virus（LYSV）,Onion yellow dwarf virus（OYDV）,and allexiviruses,with amplicon size of 592,431,338,265,and 190 bp,respectively. Using the established multiplex RT-PCR method,virus detection and analysis were conducted on 24 garlic tissue cultured plantlets and 48 field samples.【Results】The specificity of the detection system was verified by single/multiplex PCR and sequence alignment. By adjusting the annealing temperature and primer ratio,the optimal amplification effect was achieved at 56.6 ℃ annealing temperature and the primer volume ratio of ShLV,GarCLV,LYSV,OYDV,and allexiviruses at 10∶8∶3∶3∶16. All 5 viruses/virus groups had a minimum detection concentration of 1.0×10² copies/μL. The virus detection results of garlic tissue cultured plantlets were rich in diversity. The field samples were infected with at least 2 viruses/virus groups,and the viruses/virus groups could be clearly distinguished.【Conclusion】The developed multiplex RT-PCR system quickly and effectively detected and differentiated the infection status of the five viruses/virus groups in garlic,including ShLV,GarCLV,LYSV,OYDV,and allexiviruses.

Key words: Garlic, Virus, Multiplex RT-PCR, Detection system

中图分类号:

S436.33

赵永强, 樊继德, 陆信娟, 刘灿玉, 张碧薇, 杨青青, 葛洁, 史新敏, 杨峰. 大蒜病毒多重RT-PCR检测体系的建立与应用[J]. 北方农业学报, 2023, 51(1): 22-30.

ZHAO Yongqiang, FAN Jide, LU Xinjuan, LIU Canyu, ZHANG Biwei, YANG Qingqing, GE Jie, SHI Xinmin, YANG Feng. Development and application of a multiplex RT-PCR detection system for garlic viruses[J]. Journal of Northern Agriculture, 2023, 51(1): 22-30.

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大蒜病毒多重RT-PCR检测体系的建立与应用

Development and application of a multiplex RT-PCR detection system for garlic viruses

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