Detection of Botrytis cinerea by loop-mediated isothermal amplification
Detection of Botrytis cinerea by loop-mediated isothermal amplification
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DOI：
10.1111/j.1472-765x.2010.02949.x
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发表时间：
2010-12-01
影响因子：
2.4
通讯作者：
Boonham, N.
中科院分区：
文献类型：
--
作者：
Tomlinson, J. A.;Dickinson, M. J.;Boonham, N.
目的：开发一种基于环介导等温扩增 (LAMP) 的灵敏、快速且简单的灰霉病检测方法，该方法适合在常规实验室环境之外使用。方法和结果：基于灰霉病菌核核糖体 DNA (rDNA) 的基因间间隔区设计了 LAMP 检测。使用从培养物中提取的 DNA 对所得检测的灵敏度和特异性进行了表征。该测定一致扩增了 65 pg 灰霉病菌 DNA。除密切相关的天竺葵葡萄孢外，未观察到与一系列其他真菌病原体的交叉反应。使用新型实时 LAMP 平台（OptiGene Genie I）可以检测受感染玫瑰花瓣中的灰霉病菌，扩增时间小于 15 分钟。结论：所开发的 LAMP 检测方法适用于快速检测受感染植物材料中的灰霉病菌。研究的意义和影响：LAMP 方法结合了基于核酸的方法的灵敏度和特异性，以及简化的设备和缩短的反应时间。这些特征使得该方法可能适合现场使用，测试结果有助于为受灰霉病影响的商品（例如切花、水果和蔬菜）的储存和加工决策提供信息。
Aims:To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop-mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting.Methods and Results:A LAMP assay was designed based on the intergenic spacer of the B. cinerea nuclear ribosomal DNA (rDNA). The resulting assay was characterized in terms of sensitivity and specificity using DNA extracted from cultures. The assay consistently amplified 65 pg B. cinerea DNA. No cross-reactivity was observed with a range of other fungal pathogens, with the exception of the closely related species Botrytis pelargonii. Use of a novel real-time LAMP platform (the OptiGene Genie I) allowed detection of B. cinerea in infected rose petals, with amplification occurring in < 15 min.Conclusions:The LAMP assay that was developed is suitable for rapid detection of B. cinerea in infected plant material.Significance and Impact of the Study:The LAMP method combines the sensitivity and specificity of nucleic acid-based methods with simplified equipment and a reduced reaction time. These features make the method potentially suitable for on-site use, where the results of testing could help to inform decisions regarding the storage and processing of commodities affected by B. cinerea, such as cut flowers, fruit and vegetables.