多肉植物红心莲茎腐病尖孢镰刀菌LAMP快速检测技术的建立

姚锦爱; 黄鹏; 陈汉鑫; 侯翔宇; 余德亿

doi:10.19303/j.issn.1008-0384.2020.08.008

多肉植物红心莲茎腐病尖孢镰刀菌LAMP快速检测技术的建立

A LAMP Assay for Rapid Detection of Stem Rot Fusarium oxysporum on Succulent Plant, Echeveria Perle von Nürnberg

摘要

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。

Abstract:
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.

HTML全文

参考文献(20)

施引文献

资源附件(0)