徐娜娜　李鹏昌　于金凤

摘要：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。

关键词：小麦纹枯病菌；SYBR GreenⅠ荧光染色法；real-time PCR

中图分类号：S435.12：Q785 文献标识号：A 文章编号：1001-4942（2014）03-0106-04

AbstractA pair of specific primers， WKF-8/WKR-8， were designed according to the DNA sequence of the internal transcribed spacers （ITS） region of Rhizoctonia cerealis AG-D fusion group. Their specificity and sensitivity were detected. A real-time polymerase chain reaction system was developed and optimized 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.

Key wordsRhizoctonia cerealis； SYBR Green Ⅰ fluorescent staining method； Real-time PCR

小麦在国家粮食安全和农业现代化发展中占有重要地位[1]。小麦纹枯病是世界范围内严重发生的小麦病害之一。我国于1973年发现小麦纹枯病，近年来，随着小麦品种、栽培制度、肥水条件等的改变，纹枯病发生危害逐渐加重，已成为影响小麦高产、稳产的重大障碍[2]。陈莹、贾廷祥等的研究表明我国小麦纹枯病的优势致病群为AG-D融合群[3，4]。综合考虑和分析灾害对小麦产量的影响是小麦生产灾害诊断与预防的关键[5]。

目前对土壤中小麦纹枯病菌定量检测的方法主要是传统的活体分离、培养获得菌株或者使用土壤浸出液涂板，促使土壤中的病原菌形成肉眼可见的单菌落，从而估计出土壤中禾谷丝核菌的量。传统检测方法受人为影响大，可重复性低，难以实现标准化检测。近年来分子生物学的快速发展使得对病害的早期快速检测成为可能，并开始应用于纹枯病菌的早期诊断。张廷婷[6]于2012年采用荧光定量PCR 技术检测了花生种皮抗黄曲霉基因表达强度。……