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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。
One pair of primers and one Taq Man-MGB probe were designed and synthesized based on the sequence differences of Verticillium albo-atrum and its similar β-tubulin gene. Real-time fluorescence PCR detection of bacteria. The experiments on the tested strains of Rhizoctonia solani and its approximate species showed that the method was specific and only Verticillium albus could be detected. The optimized reaction conditions were as follows: the final concentration of primer was 1.0μmol / L and the final concentration of probe was 0.7μmol / L. Sensitivity test results show that the minimum detection limit for the total DNA content of 10 pg (20μL reaction system). This method is fast and sensitive, providing an important reference for the rapid detection of Verticillium albus.