葡萄卷叶病毒Ⅲ RT-PCR检测技术研究Detection of Grapevine Leafroll-associated Virus Ⅲ by Reverse Tran-scription-polymerase Chain Reaction
刘三军,郭卫东,张秋叶,何水涛,周厚成,张亚冰
摘要(Abstract)：
利用ＲＴ－ＰＣＲ技术,对葡萄卷叶病毒株系Ⅲ进行了检测。采用的技术流程为:通过提取葡萄叶片或韧皮部总ＲＮＡ,获得病毒的ＲＮＡ,反转录合成ｃＤＮＡ,经ＰＣＲ扩增,获得病毒ｃＤＮＡ的特征片段,并进行了序列分析。对提取的总ＲＮＡ进行了完整性和纯度检测,确定了良好的ＲＮＡ获得方法。结果表明,通过ＲＴ－ＰＣＲ扩增的片段序列与病毒源序列的同源性高达９９．３%,说明采用本研究确定的方法检测葡萄卷叶病毒株系Ⅲ结果可靠。
关键词(KeyWords)： 葡萄卷叶病毒Ⅲ;RT－PCR;检测;克隆;序列分析
基金项目(Foundation): 中国农业科学院院长基金资助项目(编号9921－9)及河南省重大科技攻关项目“苹果、葡萄、草莓脱毒快繁及产业化应用”资助项目
作者(Author): 刘三军,郭卫东,张秋叶,何水涛,周厚成,张亚冰
DOI: 10.13925/j.cnki.gsxb.2002.01.005
参考文献(References)：
- [1] 郭德银.葡萄病毒和类似病毒病研究进展[J].果树科学,1989,6(3):183－188.
- [2] MaeKenzie DJ, Johnson RC, Warner C. Incidence of four important viral pathogens in Canadian vineyards[J]. Plant Dis, 1996,80:955-958.
- [3] Bovey R, Martelli GP. Directory of major virus and virus-like diseases of grapevines.mediterranean fruit improvement council & international council for the study of virus and virus diseases of the grapevine [M].Bari, Italy, 1992.
- [4]Habili N,Fazeli CF,Ewart A,et al.Natural spread and molecular analysis of grapevine leafroll-associated virus 3 in Australia[J]. Phy-topathology, 1995, 85:1 418-1 422.
- [5] Minafra A , Hadidi A. Sensitive detention of grapeving virus A , B or leafroll-associated Ⅲ from viruliferous mealybugs and infacted tis-sue by cDNA amplification[J]. J Virol Methods, 1994,47:175-188.
- [6] MacKenzie DJ , McLean MA , Mukerji S , et al. Improve RNA extrac-tion from woody plant for the detection of viral pathogens by reverse transcription -polymerase chain reaction [J]. Plant Dis,1997,81:222-226.
- [7] 萨姆布鲁克.分子克隆实验指南(第二版)[M].金冬雁,黎孟枫译．北京:科学出版社,1996
- [8] Fazeli CF. Drect submission to NCBI sequence viewer, submitted(05-MAR-1995) C.F.Fazeli, Division of Horticulture, CSIRO.GPO Box 350 , Adelaide , SA5001 , Australia , 1995.
- [9] Kummert J, Marinho VLA .Rufflard G,et al. Sensitive detection of apple stem grooving and apple stem pitting virus from infected apple trees by RT-PCR[J]. Acta Horticulturae , 1998 , 472:97-104.
- [10] Daniels J, Marinho VLA , Kummert J , et al. Developmen t of colorimetric RT-PCR tests for apple stem grooving virus detection in apple trees[J]. Acta Horticulturae , 1998 , 472: 105-111.
- [11]张满良,朱水芳,赵冬兰.葡萄卷叶伴随病毒Ⅲ基因的克隆及序列分析[J]．西北农业大学学报,2000,28(5):70－75．