基于TaqMan MGB探针的葡萄茎枯病菌实时荧光PCR检测方法

 葡萄茎枯病菌是我国进境植物检疫性有害生物。带菌植物材料是病害传播的重要载体,准确、灵敏、快速的检测方法是严格执行口岸检疫措施及研究病害防控措施的有力工具。根据葡萄茎枯病菌及其近似种的细胞骨架蛋白(Actin)基因序列差异,设计并合成1对引物和1条特异性TaqMan-MGB探针,建立了葡萄茎枯病菌的实时荧光PCR检测方法。通过对反应体系的优化,确定了葡萄茎枯病菌的实时荧光PCR最佳反应条件:引物终浓度为0.6 μmol·L^-1,探针终浓度为0.6 μmol·L^-1。灵敏度试验结果显示,最低检测限为总DNA含量20 pg(20 μL反应体系)。此方法快速灵敏,整个反应1 h即可完成,检测过程完全闭管,无需PCR产物后续处理,为快速检测葡萄茎枯病菌提供了重要参考。该方法用于口岸疑似菌株检测,可成功检测出葡萄茎枯病菌。本研究建立的基于TaqMan MGB探针的荧光定量PCR检测方法为葡萄茎枯病菌的早期快速检测监测提供了有力工具。

TaqMan MGB-based real-time PCR method for the detection of Didymella glomerata

The pathogen Didymella glomerata is included in the list of Chinese quarantine pests. The diseased plant materials are the important carrier of organism for the disease transmission. The accurate, sensitive and rapid detection method at ports of entry and land border in comprehensive quarantine system would be the powerful measure for the disease prevention and control. In this study, a pair of primers and a TaqMan-MGB probe based on the actin gene sequence of D. glomerata and its similar species were designed and synthesized for real-time fluorescent PCR assay. The optimal primer concentration and probe concentration were 0.6 μmol·L^-1 and 0.6 μmol·L^-1, respectively. The detection limit of this method was 20 pg of total DNA in 20 μL reaction mixture. The real-time PCR method was rapid, sensitive and completed within a single tube, without post-PCR handling of the amplification products, which provides a valuable tool for early rapid detection and identification of D. glomerata.