葡萄茎痘伴随病毒cDNA探针检测技术研究  

Studies on cDNA Probe Detection Technology of Grapevine Rupestris Stem Pitting Associated Virus

作　　者：牛建新[1] 李西平[1] 张强[1] 赵英[1] 马兵钢[1] 

机构地区：[1]石河子大学农学院园艺系

出　　处：《西北农业学报》2007年第3期239-243,共5页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家自然科学基金资助项目(30060053;30360066);兵团科委项目(NKB02SDXNK01SW);国家科技攻关计划引导项目(2003BA546C)

摘　　要：选取经RT-PCR检测确认带葡萄茎痘伴随病毒的全球红葡萄品种,以葡萄叶片、皮层作为试材,采用SDS法提取高质量总RNA作为模板,以GRSPaV的特异互补引物引导,反转录合成cDNA,通过PCR扩增,获得830 bp的预期目的片段,并将其克隆测序,其同源性与NCBI中的相关序列的同源性达96%。利用克隆质粒合成了生物素标记的GRSPaV的cDNA探针,在此基础上,研究了影响GRSPaV斑点杂交的因素,结果表明当探针浓度为200 ng/mL,甲酰胺浓度为45%,42℃杂交反应4 h,可获得较高灵敏度。The purpose of this study want to establish a rapid, sensitive, accrual and practical detection system for Grapevine Rupestris Stem Pitting associated Virus based on internal control. These grapevine varieties such as ＂Red Golbe＂ that were detected to carry Grapevine rupestris stem pitting assoctiated virus by RT-PCT were selected as testing material. Using SDS method to extract high quality total RNA from grape leaves and phloem, which was template to synthesize the cDNA by the guide of GRSPaV special antisense primer. After that, through PCR amplification, the 830 bp special fragment was gained. According to the published GRSPaV array ,the special primer was designed and synthesized. The 830 bp special product was amplified with the method of RT-PCR, which was cloned and sequenced. The sequenced result showed the 830 bp special fragment had 96% similarity with related arrays in NCBI. Using cloned plasmid to synthesize GRSPaV＇s non-radioactive cDNA probe, on this bases, though researching the factors affecting GRSPaV spot-blotting, Studies indicated that the higher sensitivity was obtained at probe concentration 200 ng/mL, 45%formamide and 42℃ for 4 hours.

关 键 词：葡萄茎痘伴随病毒 CDNA探针 检测 

分 类 号：S436.631[农业科学—农业昆虫与害虫防治]