啤酒花潜隐类病毒的实时荧光RT-PCR检测  

Detection of Hop latent viroid by real-time fluorescent RT-PCR assay

作　　者：郭立新[1] 段维军[2] 张祥林[3] 邓丛良[4] 闻伟刚[2] 李世访[5] 

机构地区：[1]北仑出入境检验检疫局,宁波315800 [2]宁波出入境检验检疫局,宁波315012 [3]新疆出入境检验检疫局,乌鲁木齐830063 [4]北京出入境检验检疫局,北京101312 [5]中国农业科学院植物保护研究所,北京100193

出　　处：《植物病理学报》2012年第5期466-473,共8页Acta Phytopathologica Sinica

基　　金：国家质检总局课题(2009IK256;2011IK169);国家"973"课题(2011CB932800);宁波检验检疫局科研项目(甬K07-2011);国家标准制修订计划项目(20100455-T-469)

摘　　要：根据啤酒花潜隐类病毒(HpLVd)各分离物基因序列,设计并合成1对特异性引物和1条TaqMan-MGB探针,建立了对HpLVd的实时荧光RT-PCR检测方法。通过对反应体系的优化,确定了HpLVd的实时荧光RT-PCR最佳反应条件:Mg2+终浓度为5.5 mmol/L,引物终浓度为0.7μmol/L,探针终浓度为0.5μmol/L。灵敏度对比试验结果显示,实时荧光RT-PCR的灵敏度较普通RT-PCR通过琼脂糖凝胶电泳高10倍,在25μL反应体系中,总RNA含量达到20 pg就能通过实时荧光RT-PCR检测出来。此方法也能成功检测田间样品上的啤酒花潜隐类病毒。A pair of primers and a TaqMan-MGB probe based on the conserved nucleotide sequence of seve- ral Hop latent viroid （HpLVd） isolates were designed and synthesized. A novel real-time fluorescent RT-PCR was established to detect HpLVd. Optimal Mg^2＋ , primer, and probe concentration were 5.5 mmol/L, 0.7 μmol/L, and 0.5 μmol/L, respectively by optimization of real-time fluorescent RT-PCR reaction system. The method, detecting as little as 20 pg of total RNA in 25 μL reaction mixture, was ten times more sensitive than the normal RT-PCR. The real-time RT-PCR assay was applied successfully to detect HpLVd in field samples.

关 键 词：啤酒花潜隐类病毒 实时荧光RT-PCR 检测 TAQMAN-MGB探针 

分 类 号：S432.41[农业科学—植物病理学]