Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Beijing 100193, PR China.
CIMMYT Yunnan Office/Institute of Food Crops, Yunnan Academy of Agricultural Sciences, No. 2238, Beijing Road, Kunming 650200, PR China.
Int J Food Microbiol. 2019 Feb 16;291:72-78. doi: 10.1016/j.ijfoodmicro.2018.11.021. Epub 2018 Nov 20.
Fusarium temperatum is an emerging maize pathogen that causes maize ear and stalk rot diseases and produces various mycotoxins including moniliformin, beauvericin, enniatins and fumonisin B1, which poses a potential risk to the human food or animal feed supply chains. Early detection of F. temperatum is crucial to prevent its derived mycotoxins from entering the food chain, and is also a useful tool in disease management practices. Here, we describe a loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of F. temperatum. The 28S ribosomal DNA sequences (28S rDNA) of F. temperatum were used to design a set of six primers. The reaction conditions were optimized for developing a fast assay with high specificity and sensitivity, and were able to detect the presence of less than 10 pg of target DNA per reaction within 60 min. Furthermore, the resulting amplicons were visualized by adding SYBR Green I to the reaction tubes. Suspected F. temperatum infected maize stalk samples collected from Yunnan province, China were identified using the developed LAMP assay. In conclusion, the method not only provides a rapid and specific screening for the existence of F. temperatum in a bulk of maize samples without using sophisticated equipment, but also is potentially useful for other agriculturally important toxigenic fungi.
扩展青霉是一种新兴的玉米病原体，可引起玉米穗和茎腐病，并产生多种霉菌毒素，包括单端孢霉烯族毒素、伏马菌素 B1、 beauvericin 和 enniatins，这对人类食品或动物饲料供应链构成了潜在风险。早期检测扩展青霉对于防止其衍生霉菌毒素进入食物链至关重要，也是疾病管理实践中的有用工具。在这里，我们描述了一种用于快速诊断扩展青霉的环介导等温扩增（LAMP）检测方法。使用扩展青霉的 28S 核糖体 DNA 序列（28S rDNA）设计了一组 6 个引物。优化了反应条件，以开发一种快速、高特异性和高灵敏度的检测方法，能够在 60 分钟内检测到每个反应中少于 10pg 的目标 DNA。此外，通过向反应管中添加 SYBR Green I 可以可视化得到的扩增产物。使用开发的 LAMP 检测方法鉴定了从中国云南省采集的疑似扩展青霉感染的玉米茎样本。总之，该方法不仅提供了一种快速、特异性的筛选方法，用于在不使用复杂设备的情况下筛选大量玉米样品中是否存在扩展青霉，而且对于其他农业上重要的产毒真菌也具有潜在的应用价值。
