Rapid detection by a loop -mediated isothermal amplification assay based on EF-1 alpha gene for stem rot on Cymbidium ensifolium

文献类型： 外文期刊

第一作者： Yao, Jin-ai

作者： Yao, Jin-ai;Huang, Peng;Hou, Xiang-yu;Yu, De-yi

作者机构：

关键词： Cymbidium ensifolium; Fusarium oxysporum; Polymerase Chain Reaction; loop-mediated isothermal amplification; pathogen detection

期刊名称：EUROPEAN JOURNAL OF HORTICULTURAL SCIENCE （ 影响因子：1.482； 五年影响因子：1.503 ）

ISSN： 1611-4426

年卷期： 2021 年 86 卷 2 期

页码：

收录情况： SCI

摘要： Based on the Elongation factor la (EF-1 alpha) sequence of Fusarium oxysporum, a set of four LAMP (Loopmediated isothermal amplification) primers were designed, and a rapid, simple and sensitive method for the visualization detection of Fusarium oxysporum of stem rot on Cymbidium ensifolium established. The template DNA from infected stems were used for a one-step LAMP which was carried out under different temperature and time conditions. The optimal temperature and duration were found to be 64 degrees C for 60 min by SYBR Green I as a fluorescence indicator and electrophoresis in agarose gels for detection that with LAMP products in this study LAMP assay was used for specific evaluation of all strains, only F oxysporum of Cymbidium ensifolium turned yellow-green, and the other strains were all orange, including the other diseases on Cymbidium. The detection limit of E oxysporum by LAMP was 1 pg genomic DNA per 25 ILL reaction, while the limit of detection of routine polymerase chain reaction (PCR) was 10 pg. The positive ratios of LAMP, PCR, and traditional tissue isolation for detecting F oxysporum of stem rot samples with typical symptoms in Cymbidium ensifolium were 35/35 (100%), 31/35 (88.6%) and 29/35 (82.8%), respectively. In conclusion, the establishment of LAMP assay provides a new technical platform for the real-time monitoring and detection for stem rot on Cymbidium ensifolium. It meets the need of rapid detection, diagnosis, and real-time monitoring of the pathogen in production.

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