基于RPA恒温扩增技术对大豆尖孢镰刀菌的快速检测

Rapid detection of Fusarium oxysporum on soybean using recombinase polymerase amplification

尖孢镰刀菌侵染大豆引起枯萎病,导致大豆严重减产。为实现对大豆上尖孢镰刀菌的快速检测,基于重组酶聚合酶扩增(RPA)技术开展了试验研究,根据大豆尖孢镰刀菌的特异基因设计了正反引物各3个,并通过两两组合筛选出最佳引物对,实现了在恒温(39℃)条件下,反应15~20 min,即可检测90 fg/μL以上浓度的尖孢镰刀菌,且与大豆疫霉菌、大豆灰斑菌等均无交叉反应。本研究建立的大豆尖孢镰刀菌检测法,相较于传统PCR法和实时定量PCR法具有快速、便捷、即时观察的特点,为该病原的快速检测提供了新的技术手段。

The objective of this study is to rapidly detect Fusarium oxysporum on soybeans using recombinase polymerase amplification(RPA)technology through experimental research.Three forward and reverse primers were designed for the specific genes of F.oxysporum,and the optimal primer pairs were selected that can achieve a lower limit of detection at the femtogram level(90 fg/μL)under constant temperature(39°C)for 15 to 20 minutes.There was no cross-reaction with Phytophthora megasperma f.sp.glycinea and Cercospora sojina.Compared to traditional PCR and real-time quantitative PCR,the RPA detection method for Fusarium in soybeans,established in this study,is rapid,convenient,and allows for immediate observation.This provides a new technical means for the rapid detection of this pathogen.