葡萄斑点病毒的RT-PCR检测  

RT-PCR detection of Grapevine fleck virus

作　　者：卓娜[1] 王国平[1] 邓丛良[2] 洪霓[1] 

机构地区：[1]华中农业大学植物科学技术学院,武汉430070 [2]北京出入境检验检疫局植物检疫中心,北京100026

出　　处：《果树学报》2011年第4期717-720,共4页Journal of Fruit Science

基　　金：国家"十一五"支撑计划项目(2006BAD08A16);国家公益性课题(20111035)

摘　　要：由葡萄斑点病毒(Grapevine fleck virus,GFkV)引起的葡萄斑点病是世界上普遍发生的葡萄病毒病害。采用RT-PCR从表现明显斑点症状的葡萄样品维多利亚、无核白鸡心和胜宝叶片中检测到预期大小为179 bp的片段。对这些扩增片段进行克隆、序列测定及比对分析,结果表明,来源于这3个样品的GFkV序列间相似性为97.2%～98.9%,与NCBI已登录GFkV序列AJ309022的相似性为96.6%～97.8%。在此基础上,采用纳米磁珠法从以上3个葡萄品种的休眠枝条韧皮部与休眠芽中提取病毒RNA,根据上述已测序列设计合成了TaqMan探针与引物,初步建立了GFkV的MNP Real time-RT-PCR检测体系。结果表明,无核白鸡心中GFkV含量最高,且该病毒在休眠芽中的含量比韧皮部约高出10倍,通过MNP Real time-RT-PCR最低可从100μg葡萄组织中检测到GFkV。Grapevine fleck is a common grapevine virus disease in the world,which is caused by Grapevine fleck virus（GFkV）.A pair of primers F1/R1 were synthesized based on the reported GFkV sequence,and the products with expected 179 bp were amplified by RT-PCR from the leaves of grapevine samples Centennial seedless,Victoria and Shengbao which showed fleck symptoms.These products were cloned and sequenced.Sequence comparison showed that the similarities among sequences from the three samples were 97.2% to 98.9% and shared 96.6% to 97.8% similarities with the reported sequence（GeneBank accession no.AJ309022）.The virus RNAs were extracted from buds and phloem tissues of the dormant grapevine canes by magnetic nano-particles（MNP） method,and the TaqMan probe and primers were designed based on the above sequences.The MNP RT-PCR method was primarily established for the detection of GFkV.The results showed that the GFkV titers were relatively higher in Centennial seedless than in the other two samples,and it was 10 times higher in dormant buds than in phloem.The detection sensitivity for GFkV by MNP-Real time-RT-PCR was about 100 μg grapevine tissues.

关 键 词：葡萄斑点病毒 纳米磁珠 Realtime-RT-PCR 

分 类 号：S663.1[农业科学—果树学]