Technische Universität München, Lehrstuhl für Technische Mikrobiologie, Freising, Germany.
Int J Food Microbiol. 2012 Jun 1;156(3):189-96. doi: 10.1016/j.ijfoodmicro.2012.03.009. Epub 2012 Mar 16.
Fusarium graminearum and the closely related F. culmorum were found to be associated with over foaming of bottled beer (gushing) when contaminated brewing malt is used. The presence of highly surface active hydrophobins produced by these fungi upon growth on wheat or barley in the field or during malting may affect bubble formation and stability in gushing beers and other carbonated beverages. Aiming for a method for the rapid and user friendly analysis of unmalted and malted cereals during quality control in the brewing industry, a loop-mediated isothermal amplification (LAMP) assay for the detection of Fusarium spp. capable of producing the gushing inducing hydrophobin Hyd5p was set up. A set of primers was designed towards a 221 bp region within the hyd5 gene of F. culmorum. The LAMP product was verified by sequencing a 150 bp portion. Testing specificity with purified DNA from 99 different fungal species as well as barley and wheat showed that DNA synthesis only occurred during LAMP when DNA of the closely related species F. graminearum, F. culmorum, F. cerealis and F. lunulosporum were used as template. In-tube indirect detection of DNA amplification was applied using manganese-quenched calcein as fluorescence indicator for pyrophosphate produced during DNA synthesis. The assay had a detection limit of 0.74 pg of purified target DNA which corresponds 20 copy numbers per reaction within 30 minutes using a simple heating block. Analysis of Fusarium infected cereals revealed that the assay was able to detect F. graminearum at a level of 0.5% of infected grains in uninfected barley by analysis of surface washings without further sample preparation. Results show that the hyd5 based LAMP assay can be a rapid, useful and sensitive tool for quality control in the brewing and malting industry.
镰刀菌属和与其密切相关的禾谷镰刀菌被发现与使用污染酿造麦芽时的瓶装啤酒（喷涌）过度发泡有关。当这些真菌在田间或麦芽生产过程中在小麦或大麦上生长时，会产生具有高度表面活性的疏水蛋白，这可能会影响喷涌啤酒和其他碳酸饮料中的气泡形成和稳定性。为了在酿造行业的质量控制期间对未发芽和发芽谷物进行快速且用户友好的分析，建立了一种用于检测能够产生诱导喷涌的疏水蛋白 Hyd5p 的镰刀菌属的环介导等温扩增（LAMP）检测方法。针对 F. culmorum 的 hyd5 基因内的 221 bp 区域设计了一组引物。通过对 150 bp 部分进行测序来验证 LAMP 产物。用来自 99 种不同真菌物种以及大麦和小麦的纯化 DNA 进行特异性测试表明，只有当使用密切相关的物种禾谷镰刀菌、禾谷镰刀菌、禾谷镰刀菌和旋孢镰刀菌的 DNA 作为模板时，LAMP 才会发生 DNA 合成。通过使用锰猝灭钙黄绿素作为 DNA 合成过程中产生的焦磷酸的荧光指示剂，在管内间接检测 DNA 扩增。该测定法的检测限为 0.74 pg 纯化的靶 DNA，使用简单的加热块在 30 分钟内每个反应的检测限为 20 个拷贝数。对感染镰刀菌的谷物进行分析表明，该测定法能够在未受感染的大麦中以 0.5%感染谷物的水平检测到禾谷镰刀菌，而无需进一步的样品制备即可对表面冲洗液进行分析。结果表明，基于 hyd5 的 LAMP 测定法可以成为酿造和麦芽行业质量控制的快速、有用和敏感工具。
