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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 specific primers and a TaqMan-MGB probe were designed and synthesized according to the sequence of HpLVd isolates. A real-time fluorescent RT-PCR method was established for HpLVd. The optimum reaction conditions for real-time fluorescence RT-PCR of HpLVd were determined by optimizing the reaction system. The final concentration of Mg2 + was 5.5 mmol / L, the final concentration of primer was 0.7μmol / L, and the final concentration of probe was 0.5μmol / L. Sensitivity comparison test results show that real-time fluorescence RT-PCR sensitivity than ordinary RT-PCR by agarose gel electrophoresis 10 times higher in 25μL reaction system, the total RNA content of 20 pg can be detected by real-time fluorescence RT-PCR . This method can also successfully detect hop latent virus on field samples.