燕麦镰孢菌环介导等温扩增(LAMP)检测方法的建立与应用

Development of a Loop-mediated Isothermal Amplification Assay for Detection of Fusarium avenaceum

为了明确燕麦镰孢菌(Fusarium avenaceum)快速检测方法,以荧光染料SYBR Green I为指示剂,以燕麦镰孢菌(F.avenaceum)的ITS序列为目的 DNA片段,应用LAMP设计软件设计4条引物,优化LAMP反应体系和反应条件,用2%琼脂糖凝胶电泳和LAMP反应液颜色变化进行特异性和灵敏度验证,同时进行田间发病组织检测和土壤燕麦镰孢菌灵敏度验证。结果表明:优化的LAMP反应体系,最佳反应温度为65℃,反应程序为65℃下1h,80℃下20min。特异性检测结果表明,燕麦镰孢菌LAMP检测均呈黄绿色(阳性),对照和其他病原菌均呈橘色(阴性)。灵敏度验证结果表明,LAMP反应液检测灵敏度在DNA水平达到10pg·μL-1,每克土壤中检测的灵敏度为40个燕麦镰孢菌孢子。对采自甘肃省甘南州卓尼县和临潭县的20份疑似病害样本提取的DNA进行检测,其中13份为阳性,表明该技术能够有效的检测出青稞发病组织中的燕麦镰孢菌。本方法的建立与应用能为燕麦镰孢菌的检测及其青稞茎基腐病的诊断提供一种新技术。

In order to determine Fusarium avenaceum with rapid method,a loop-mediated isothermal amplification(LAMP)was first developed using SYBR Green I as a fluorescence indicator in this study.Based on the internal transcribed spacer(ITS)sequence of DNA genes in F.avenaceum,a set of four LAMP primers were designed using software.The reaction systems and conditions were optimized.The specificity and sensitivity of LAMP were assayed by electrophoresis on 2.0%agarose gel and SYBR Green I stain.Diseased naked barley tissue and F.avenaceum spores in soil were evaluated.The results showed that the LAMP assay efficiently amplified the target DNA in 1 h at 65℃and 20 min at 80℃with the optimum reaction temperature at 65℃.A positive yellow green color was only observed in the presence of F.avenaceum,whereas all other isolates showed orange color as negative control.The detection limit of the LAMP assay for F.avenaceum was 10 pg·μL^-1 of target DNA or 40 spores in 1 g soil in the high sensitivity.The diagnostic validation was performed to detect F.avenaceum in 13 out of 20 suspect diseased naked barley samples collected from Lintan and Zhuoni County in Gannan state,Gansu province.This study provided a new approach for the detection of F.avenaceum and naked barley crown rot disease.