应用real-time PCR定量检测土壤中小麦纹枯病菌方法的建立

Quantitative Detection of Rhizoctonia cerealis in Soil by Real-Time PCR

根据小麦纹枯病菌Rhizoctonia cerealis AG-D融合群ITS区的DNA序列设计特异性引物对WKF-8/WKR-8，对引物的特异性和灵敏性进行检测，建立并优化基于 SYBR GreenⅠ荧光染料法的 real -time PCR反应体系，绘制标准曲线。检测范围在1．0×10-3～10 ng/μl之间有良好的线性关系，相关系数R2为0．995，扩增效率为99．7％，灵敏度比常规PCR方法高100倍。结果表明，该方法具有快速、特异性强、敏感度高等特点。

A pair of specific primers,WKF -8/WKR -8,were designed according to the DNA se-quence of the internal transcribed spacers （ITS）region of Rhizoctonia cerealis AG-D fusion group.Their spe-cificity and sensitivity were detected.A real-time polymerase chain reaction system was developed and opti-mized based on SYBR GreenⅠfluorescent staining method.And the standard curve was drawn.A good linear relationship between the template DNA amount and cycle threshold （Ct）value was observed in the range of 1.0 ×10 -3 ^10 ng/μl.The correlation coefficient was 0.995,and the amplification efficiency was 99.7%. The sensitivity was 100 times higher than that of conventional PCR.The results showed that this method had characteristics of speediness,high sensitivity and specificity.