应用实时荧光PCR检测甜菜霜霉病菌

Detection of Peronospora farinosa sp.betea Byford by Real-time PCR

【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 is to develop a SYBR GreenⅠreal-time PCR for the detection of Peronospora farinosa f. sp. betea Byford rapidly.【Method】The real-time PCR primers were designed based on the reported 28 S r DNA gene sequences of P. farinosa sp. betea Byford and similar species.Totally 6 pathogens of beet and other 5 pathogens and 12 samples of beet seeds were screened to test the specificity and sensitivity and applicability in this assay.【Result】The SYBR GreenⅠreal-time PCR assay could detect positive amplification from P. farinosa f. sp. betea Byford only,negative results from other pathogens and negative control,and more than 100 fg genomic DNA of P. farinosa sp. betea Byford could be detected.The 12 samples of beet seeds from different sources were detected by this assay,suggesting that the results were consistent with the field monitoring results.【Conclusion】This assay can detect P. farinosa sp. betea Byford rapidly,and offer a technology to early diagnosis and control P. farinosa sp. betea Byford.