【摘要】 根据黑白轮枝菌(Verticillium albo-atrum)及其近似种β-微管蛋白基因(β-tubulin)序列差异,设计并合成1对引物和1条Taq Man-MGB探针,建立了黑白轮枝菌的实时荧光PCR检测方法。对供试黑白轮枝菌及其近似种实验表明,该方法特异性强,只有黑白轮枝菌可被检出。通过对反应体系的优化,确定了最佳反应条件:引物终浓度为1.0μmol/L,探针终浓度为0.7μmol/L。灵敏度试验结果显示,最低检测限量为总DNA含量10 pg(20μL反应体系)。此方法快速灵敏,为快速检测黑白轮枝菌提供了重要参考。更多还原

【Abstract】 A pair of primers and a Taq Man-MGB probe based on the beta-tubulin sequence of V. albo-atrum and related isolates were designed and synthesized. A real-time fluorescent PCR was established to detect V. albo-atrum. The assay was validated with V. albo-atrum,it’s phylogenetically most closely related species. Optimal primer concentration and probe concentration were 1.0 μmol/L and 0.7 μmol/L,respectively.The method detected as little as 10 pg of total DNA in 20 μL reaction mixture. The method was rapid,sensitive and completed within a single tube,without post-PCR handling of the amplification products. The new method provides a valuable tool for early rapid detection and identification of V. albo-atrum.更多还原