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采用高通量实时荧光定量PCR技术建立检测和监测油菜菌核病菌群体数量的方法。利用油菜菌核病菌(Sclerotiniasclerotiorum)的β-微管蛋白基因内含子序列的特异性,设计引物对SclSF(5’-CTCAAATCTCCGAAAGTT-3’)/SclAF(5’-TGCAGACGGGTAATATG-3’),建立和优化了SYBR GreenⅠ实时荧光定量PCR检测体系。结果表明,该引物对能够从8种所测试的十字花科植物常见病原真菌中特异性扩增出油菜菌核病菌;所建立的实时定量PCR技术可应用于油菜病叶和病茎中菌核病菌的早期检测及菌核病的预测预报。
A high-throughput real-time PCR method was established to detect and monitor the population of rape Sclerotinia. The primer pair SclSF (5’-CTCAAATCTCCGAAAGTT-3 ’) / SclAF (5’-TGCAGACGGGTAATATG-3’) was designed using the specificity of the β-tubulin gene intron sequences of Sclerotinasclerotiorum to establish and Optimized SYBR Green Ⅰ real-time fluorescence quantitative PCR detection system. The results showed that this primer pair could specifically amplify Sclerotinia sclerotiorum from 8 kinds of common pathogenic fungi tested in cruciferous plants. The established real-time quantitative PCR technique could be applied to the sclerotia of diseased rapeseed and diseased stem Early Detection of Bacteria and Prediction of Sclerotinia.