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【目的】建立用于快速检测甜菜霜霉病菌Peronospora farinosa f.sp.betea Byford的SYBR GreenⅠ实时荧光PCR检测方法。【方法】根据已报道的P.farinosa f.sp.Betea Byford及近似种28S r DNA序列同源性比对结果,设计用于检测甜菜霜霉病菌的SYBR GreenⅠ实时荧光PCR检测引物。利用所建立的方法对包括P.farinosa f.sp.Betea在内的6种甜菜上的病原菌和5种其他霜霉病菌基因组DNA及12份甜菜种子样品进行检测,验证该方法的检测特异性、灵敏度及实用性。【结果】所建立的检测方法仅对甜菜霜霉病菌得到阳性扩增,其它参照菌株及阴性对照均无荧光信号扩增,准确排除了甜菜上其他5种病菌的干扰,其检测灵敏度显示最低限量为100 fg病菌DNA,应用该方法对不同来源的12份甜菜种子样品进行检测,田间监测结果一致。【结论】所建立的荧光检测方法能够对甜菜霜霉病菌进行快速筛查,为甜菜霜霉病菌的早期诊断和防控提供了一种技术手段。
【Objective】 The objective of this study was to establish a SYBR Green Ⅰ real-time PCR assay for the rapid determination of Peronospora farinosa f. Sp. Betea Byford. 【Method】 SYBR Green Ⅰ real-time PCR primers were designed to detect the mycorrhizal fungi in P.farinosa f.sp.Betea Byford and the similar 28S r DNA sequences. The established method was used to detect the pathogenic bacteria in 6 beet species including P.farinosa f. Sp. Bettea and 5 other Genomic DNA of downy mildew and 12 beet seed samples to verify the detection specificity of this method, Sensitivity and practicality. 【Result】 The results showed that the method established only positive amplification for the fungus Beetown fungus, no fluorescence signal amplification was found in other reference strains and negative control, which accurately excluded the interference of other five kinds of bacteria on beet, the detection sensitivity showed the lowest limit For 100 fg of bacterial DNA, 12 beet seed samples from different sources were detected by this method. The field monitoring results were consistent. 【Conclusion】 The established fluorescence detection method can be used for rapid screening of beet fungi, which provides a technical measure for the early diagnosis and control of beet fungus.