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。
A pair of primers and a TaqMan-MGB probe based on the beta-tubulin sequence of V. albo-a- trum and related isolates were designed and synthesized. A real-time fluorescent PCR was established to de- tect 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,sen- sitive 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.
出处
《植物检疫》
北大核心
2017年第1期32-36,共5页
Plant Quarantine
基金
国家重点研发计划(2016YFF0203201)
浙江省博士后择优资助项目(BSH1402011)
宁波市科研项目(2015C110018&2014C50082)
