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.
出处
《植物病理学报》
CAS
CSCD
北大核心
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)
