乐都紫皮大蒜4种病毒多重RT-PCR检测技术的建立  

Establishment of a multiplex RT-PCR system for simultaneous detection of four viruses in Ledu purple garlic

作　　者：张超 董怡衡 岳苗 卫唯 张谦 王乐 尹卫 王煜伟 梁健 ZHANG Chao;DONG Yiheng;YUE Miao;WEI Wei;ZHANG Qian;WANG Le;YIN Wei;WANG Yuwei;LIANG Jian(State Key Laboratory of Plateau Ecology and Agriculture,Qinghai University,Xining,Qinghai 810016,China)

机构地区：[1]青海大学省部共建三江源生态与高原农牧业国家重点实验室,青海西宁810016

出　　处：《西北农林科技大学学报（自然科学版）》2023年第1期120-129,共10页Journal of Northwest A&F University(Natural Science Edition)

基　　金：青海省科技计划项目(2019-NK-106)。

摘　　要：【目的】建立乐都紫皮大蒜4种病毒的多重RT-PCR快速检测方法,为大蒜病毒病病原鉴定、脱毒效果验证等提供技术支撑。【方法】根据乐都紫皮大蒜RNA全长转录组测序结果,分别设计洋葱黄矮病毒(onion yellow dwarf virus,OYDV)、大蒜潜隐病毒(garlic latent virus,GLV)、大蒜D病毒(garlic virus D,GarVD)和大蒜X病毒(garlic virus X,GarVX)4种病毒的外壳蛋白特异性引物,筛选不同引物,优化引物用量、模板用量、退火温度等条件,并进行了多重RT-PCR的灵敏度检测和12份引进栽培大蒜种质资源的病毒检测。【结果】通过优化筛选,在一个PCR反应体系中实现了OYDV(1232 bp)、GLV(831 bp)、GarVD(506 bp)和GarVX(116 bp)4种病毒的多重检测,最优反应条件为退火温度55℃,模板用量2.5μL,混合引物用量1.0μL。多重RT-PCR的灵敏度略低于单一RT-PCR。对引进的12份栽培大蒜资源进行4种病毒的多重RT-PCR检测发现,有5份资源存在4种病毒,4份资源存在2种病毒,3份资源存在1种病毒。对7份资源中经多重PCR未检测到的病毒,使用单一RT-PCR进行检测和验证发现,除1份内蒙古资源(NMG)的1种病毒(GarVX)外,多重RT-PCR结果与单一RT-PCR检测结果一致。【结论】本研究建立的大蒜多重RT-PCR体系可靠性强,可用于大蒜病毒的检测与防控。【Objective】A multiplex RT-PCR rapid detection method for four viruses of Ledu purple garlic was established to provide support for identification of virus pathogen and verification of elimination efficiency.【Method】According to sequencing results of the full-length transcriptome,primer sets for onion yellow dwarf virus(OYDV),garlic latent virus(GLV),garlic virus D(GarVD),and garlic virus X(GarVX)coat proteins were designed and a multiplex RT-PCR system was established.After optimizing PCR conditions including primer concentration,annealing temperature,and template concentration,the sensitivity of the multiplex RT-PCR system was tested.Reliability of the detection system was further verified by amplification tests of 12 introduced garlic germplasms.【Result】After optimization,a multiplex RT-PCR system was established for simultaneous detection of four viruses.The amplicons corresponding to fragments of virus coat proteins of OYDV,GLV,GarVD and GarVX were 1232 bp,831 bp,506 bp and 116 bp,respectively.The optimal amplification conditions included annealing temperature of 55℃,template quantity of 2.5μL,and amount of 1.0μL for each primer.The sensitivity of the multiplex RT-PCR system was slightly lower than that of single RT-PCR.Using the multiplex RT-PCR system,it was found that 5 cultivars showed concurrent infection of 4 viruses,4 cultivars showed concurrent infection of 2 viruses,and 3 cultivars showed infection of a single virus.The detection of single and two-virus-infecting samples was further confirmed by routine PCR,which showed good consistence to the multiplex RT-PCR assay except for GarVX in NMG cultivar.【Conclusion】The established garlic multiple RT-PCR system showed strong reliability and could be used for the detection,prevention and control of garlic virus.

关 键 词：乐都紫皮大蒜 病毒检测 多重RT-PCR 洋葱黄矮病毒 大蒜潜隐病毒 大蒜D病毒 大蒜X病毒 

分 类 号：S633.4[农业科学—蔬菜学] S432.41[农业科学—园艺学]