摘要
【目的】建立一种景天科多肉植物红心莲茎腐病菌尖孢镰刀菌(Fusarium oxysporum)快速、简便、灵敏的LAMP可视化检测方法,在茎腐病发生初期实现病原菌的快速准确检测,为病害的早期监测、诊断及防治提供依据。【方法】以EF-1a(Elongation factor-1α)基因序列为靶序列,设计LAMP(Loopmediated isothermal amplification)特异性引物;以红心莲尖孢镰刀菌DNA为模板,优化反应温度和反应时间,建立LAMP检测反应体系,开展特异性和灵敏度验证及田间病株检测。【结果】建立的LAMP反应体系可有效检测出多肉植物红心莲茎腐病尖孢镰刀菌,最佳反应温度、反应时间分别为65℃,60 min;特异性验证表明红心莲尖孢镰刀菌DNA呈绿色阳性反应,灵敏度验证表明LAMP检测最小质量浓度是10 fg·μL^-1,比常规PCR提高了10倍;采用LAMP法对15份疑似红心莲茎腐病田间样本进行快速检测,检出率为100%。【结论】本研究建立的多肉植物红心莲茎腐病菌LAMP快速检测方法不仅特异性强、灵敏度高,而且检测结果可视、假阳性低,适合田间尖孢镰刀菌引起的红心莲茎腐病的快速检测。
【Objective】A LAMP(loop-mediated isothermal amplification)assay for rapid,visible and sensitive detection of Fusarium oxysporum on the succulent plant,Echeveria Perle von Nürnberg,was developed for early prevention and control of the disease.【Method】Based on the elongation factor 1α(EF-1α)sequence of F.oxysporum,LAMP primers were designed.Template DNA from the infected stems were used to optimize the reaction temperature and time.Specificity and sensitivity of the assay were verified on the detection on infected plants from the field.【Result】The newly developed LAMP assay at 65°C for 60 min effectively detected F.oxysporum on Echeveria.The assay gave the positive yellowish green result only on F.oxysporum of Echeveria.It had a detection limit of 10 fg·μL^-1 and was 100%positive on detecting F.oxysporum from 15 Echeveria samples showing typical stem rot symptoms.【Conclusion】The established LAMP assay had high specificity and sensitivity with visual and low false positive results.It was considered suitable for rapid detection of F.oxysporum on Echeveria Perle von Nürnberg in the field.
作者
姚锦爱
黄鹏
陈汉鑫
侯翔宇
余德亿
YAO Jinai;HUANG Peng;CHEN Hanxin;HOU Xiangyu;YU Deyi(Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests/Institute of Plant Protection,Fujian Academy of Agricultural Sciences,Fuzhou,Fujian 350013,China;Zhangzhou Institute of Agricultural Science,Zhangzhou,Fujian 363005,China)
出处
《福建农业学报》
CAS
CSCD
北大核心
2020年第8期863-868,共6页
Fujian Journal of Agricultural Sciences
基金
福建省科技计划公益类专项(2018R1025-2)
福建省农业科学院项目(STIT2017-2-2、DEC201907)。
关键词
红心莲
茎腐病
尖孢镰刀菌
环介导等温核酸扩增技术(LAMP)
检测
Echeveria Perle von Nürnberg
stem rot
Fusarium oxysporum
loop-mediated isothermal amplification(LAMP)
detection
