【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。更多还原

【Abstract】 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 Mg2+,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.更多还原