玉米茎基腐病菌LAMP检测技术的建立

侯翔宇; 黄鹏; 张杰; 余德亿; 姚锦爱; 李本金

玉米茎基腐病菌LAMP检测技术的建立

LAMP Assay for Rapid Fusarium verticillioides Detection on Corn Plant

摘要

摘要:
目的建立一种玉米茎基腐病菌快速、简便、灵敏的环介导等温扩增（Loop-mediated isothermal amplification, LAMP）可视化检测方法，可在植株显症前或发病初期实现病害早期精准诊断和有害生物检测预警，为病害防治提供方向和依据。
方法以病原菌的EF-1a（Elongation factor-1α）基因序列为靶序列，设计了一套LAMP特异性引物；以其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反应体系可特异有效地检测出拟轮枝镰刀菌，最佳反应温度、反应时间分别为65℃、60 min；特异性验证结果表明，拟轮枝镰孢菌DNA呈黄绿色阳性反应，灵敏度验证结果表明LAMP检测最小浓度为10 fg·μL⁻¹；采用建立的LAMP检测技术对35份采自田间疑似感染玉米茎基腐病样本检测，有17份样本的LAMP检测结果均显示为拟轮枝镰孢菌阳性，18份样本显示为拟轮枝镰孢菌阴性，与组织分离法进行形态学鉴定的结果一致。
结论本研究建立的拟轮枝镰刀菌LAMP快速检测技术不仅特异性强、灵敏度高，且检测结果可视，为病害的早期监测、快速诊断和开展及时有效的防治提供依据。

Abstract:
Objective A rapid, sensitive, and visual loop-mediated isothermal amplification (LAMP)-based method to detect Fusarium verticillioides of corn stalk rot was developed.
Method A set of primers based on the Elongation factor-1α sequence of F. verticillioides with the template DNA from infected corn leaves and optimized temperature and time were applied for the LAMP assay. Specificity and sensitivity of the prototype method were tested to verify the assay applicability for accurate pathogen detection on clinic specimens.
Result Allowing a reaction time of 60m at 65℃, the assay effectively and specifically detected F. verticillioides on the diseased corn plants. It had a detection limit of 10 fg·μL⁻¹ and provided results consistent with the morphological diagnosis. On 35 field-collected corn plants that were suspected of stalk rot infection, the LAMP assay as well as the physiological identification found 17 to be F. verticillioides-positive and 18 negative.
Conclusion The newly established LAMP assay could visually, rapidly, specifically, and accurately detect F. verticillioides on the infected corn plants. It was adequate for monitoring and early diagnosis to facilitate effective control of the disease.

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