土壤中烟草根黑腐病菌的real-time PCR分子检测

Real-time PCR molecular detection of tobacco black root rot in soil

根据烟草根黑腐菌与其它近缘真菌在ITS序列上的差异,设计PCR检测引物TbF/TbR,并通过对各菌株基因组DNA扩增验证其特异性;以接种10倍浓度梯度的根黑腐菌孢子的土壤DNA为模板,建立土壤烟草根黑腐菌real-time PCR检测的标准曲线,得出每个反应体系中分生孢子数目的对数(x)和对应的Ct值(y)之间呈线性关系:y=-1.5373x+24.955,R2=0.9909(P<0.01)。通过对5块烟田土壤烟草根黑腐菌检测和烟田烟草根黑腐病的调查,证实上述方法可以准确检测土壤中烟草根黑腐菌孢子数量。

According to the difference in ITS sequence between black rot fungus of tobacco root and other relative fungi, a pair of specific primer TbF/TbR was designed, and its specificity was verified by genomic DNA amplification. The standard curve for real-time PCR detection of black rot fungi of tobacco root was established by using soil DNA inoculated with spores of black rot fungi (10 times in concentration gardient) as a template. It was found that there was a linear relationship between the logarithm (x) of number of conidia in each reaction system(x) and the corresponding Ct value(y):y=-1.5373x+24.955, R^2=0.9909 (P < 0.01). Through detection of black rot fungi of tobacco root in five tobacco fields and the investigation of black rot disease of tobacco root in tobacco field, the above method was proved to be capable of accurately detecting the number of black rot fungi of tobacco root in soil.