基于RPA的玉米锈病菌快速检测技术的研究

Development of RPA assay for detection of the corn rust pathogens Puccinia polysora Underw. and Puccinia sorghi Schw

玉米锈病是全球玉米生产的毁灭性病害之一,病菌孢子可随气流进行传播,容易引起病害的大流行,对我国玉米产量造成重大损失。本研究根据多堆柄锈菌(Puccinia polysora Underw.)和玉米柄锈菌(Puccinia sorghi Schw.)的ITS序列设计引物,建立了基于重组酶聚合酶扩增(Recombinase polymerase amplification,RPA)的检测方法,其中多堆柄锈菌RPA检测方法的灵敏度可达7.85 pg/μL,玉米柄锈菌的RPA检测方法灵敏度可达9.9 pg/μL,经过特异性验证可有效区分玉米锈病两种不同菌株,并经过田间样品进行验证,结果表明本实验所设计的两种锈菌的RPA特异性引物检测效果较好,且操作简便,为基层一线人员提供了一种能快速鉴定多堆柄锈菌和玉米柄锈菌的新方法,对我国玉米锈病的早期预警和防控具有重要意义。

Corn rust is one of the destructive diseases in global corn production,and the spores of the pathogen can spread with the airflow,easily causing a pandemic of the disease and causing significant losses to China's corn production. This study designed primers based on the ITS sequences of Puccinia polysora Underw. and Puccinia sorghi Schw., and established a detection method based on Recombinase Polymerase Amplification(RPA). The sensitivity of the RPA detection method for Puccinia polysora Underw. can reach 7.85 pg/μL. The sensitivity of the RPA detection method for Puccinia sorghi Schw.can reach 9.9 pg/μL. After specific validation,two different strains of corn rust can be effectively distinguished,and field samples have been validated. The results show that the RPA specific primers designed in this experiment have good detection effects and are easy to operate. It provides a new method for frontline personnel to quickly identify Puccinia polysora Underw. and Puccinia sorghi Schw.,which is of great significance for early warning and prevention of corn rust in China.