Technische Universität München, Lehrstuhl für Technische Mikrobiologie, Weihenstephaner Steig 16, D-85350 Freising, Germany.
Int J Food Microbiol. 2010 Jun 15;140(2-3):183-91. doi: 10.1016/j.ijfoodmicro.2010.03.036. Epub 2010 Apr 1.
Loop-mediated isothermal amplification (LAMP) of DNA is a simple, cost effective, and rapid method for the specific detection of genomic DNA using a set of six oligonucleotide primers with eight binding sites hybridizing specifically to different regions of a target gene, and a thermophilic DNA polymerase from Geobacillus stearothermophilus for DNA amplification. The method has been applied in various assays for the diagnosis of bacterial and viral infections of humans and animals, sexing of bovine and swine embryos, and in the detection of bacteria from environmental samples. Only recently, first applications for fungal organisms were published. During the current study a LAMP assay was developed for the specific detection of Fusarium graminearum, the major causative agent of Fusarium head blight of small cereals and producer of the mycotoxins deoxynivalenol, nivalenol, and zearalenone. The assay was based on the gaoA gene (galactose oxidase) of the fungus. Amplification of DNA during the reaction was indirectly detected in situ by using calcein fluorescence as a marker without the necessity of time-consuming electrophoretic analysis. The assay was optimized for rapidness, specificity, and sensitivity and was shown to detect the presence of less than 2pg of purified target DNA per reaction within 30 min. Within 132 fungal species tested, exclusively DNA isolated from cultures of F. graminearum (lineages 1-9) resulted in a fluorescent signal after amplification with the LAMP assay. The method was demonstrated to be useful in the analysis of fungal cultures by direct analysis of surface scrapings from agar plate cultures, direct testing of single infected barley grains, and detection of F. graminearum in total genomic DNA isolated from bulk samples of ground wheat grains. Results obtained indicate that LAMP offers an interesting new assay format for the rapid and specific DNA-based detection and identification of agriculturally important toxigenic fungi in pure cultures and in contaminated sample materials and therefore presents an alternative to PCR-based assays.
环介导等温扩增（LAMP）是一种简单、经济高效且快速的方法，可使用一组六个寡核苷酸引物和八个与靶基因的不同区域特异性杂交的结合位点，以及来自嗜热细菌 Geobacillus stearothermophilus 的耐热 DNA 聚合酶来特异性检测基因组 DNA。该方法已应用于各种检测，用于诊断人类和动物的细菌和病毒感染、牛和猪胚胎的性别鉴定，以及从环境样本中检测细菌。直到最近，才首次应用于真菌生物。在当前的研究中，开发了一种用于特异性检测禾谷镰刀菌（Fusarium graminearum）的 LAMP 检测方法，禾谷镰刀菌是小谷物赤霉病的主要病原体，也是脱氧雪腐镰刀菌烯醇、雪腐镰刀菌烯醇和玉米赤霉烯酮等霉菌毒素的生产者。该检测方法基于真菌的 gaoA 基因（半乳糖氧化酶）。在反应过程中，通过使用钙黄绿素荧光作为标记物，间接在原位检测 DNA 扩增，而无需耗时的电泳分析。该检测方法经过快速性、特异性和灵敏度的优化，结果表明，每个反应可在 30 分钟内检测到少于 2pg 的纯化靶 DNA。在测试的 132 种真菌中，仅当使用 LAMP 检测方法扩增时，才会从禾谷镰刀菌（谱系 1-9）的培养物中分离出的 DNA 产生荧光信号。该方法已被证明可用于通过直接分析琼脂平板培养物表面刮屑、直接测试单个受感染的大麦粒以及从磨碎的麦粒总基因组 DNA 中检测 F. graminearum 来分析真菌培养物，在纯培养物和污染样品材料中对农业上重要的产毒真菌进行快速、特异性的基于 DNA 的检测和鉴定，因此是 PCR 检测方法的替代方法。
