Bluhm B H, Flaherty J E, Cousin M A, Woloshuk C P
Department of Botany and Plant Pathology, Purdue University, 1155 Lilly Hall, West Lafayette, Indiana 47907, USA.
J Food Prot. 2002 Dec;65(12):1955-61. doi: 10.4315/0362-028x-65.12.1955.
The genus Fusarium comprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In this study, a multiplex polymerase chain reaction (PCR) assay was developed for the group-specific detection of fumonisin-producing and trichothecene-producing species of Fusarium. Primers for genus-level recognition of Fusarium spp. were designed from the internal transcribed spacer regions (ITS1 and ITS2) of rDNA. Primers for group-specific detection were designed from the TRI6 gene involved in trichothecene biosynthesis and the FUM5 gene involved in fumonisin biosynthesis. Primer specificity was determined by testing for cross-reactivity against purified genomic DNA from 43 fungal species representing 14 genera, including 9 Aspergillus spp., 9 Fusarium spp., and 10 Penicillium spp. With purified genomic DNA as a template, genus-specific recognition was observed at 10 pg per reaction; group-specific recognition occurred at 100 pg of template per reaction for the trichothecene producer Fusarium graminearum and at 1 ng of template per reaction for the fumonisin producer Fusarium verticillioides. For the application of the PCR assay, a protocol was developed to isolate fungal DNA from cornmeal. The detection of F. graminearum and its differentiation from F. verticillioides were accomplished prior to visible fungal growth at <10(5) CFU/g of cornmeal. This level of detection is comparable to those of other methods such as enzyme-linked immunosorbent assay, and the assay described here can be used in the food industry's effort to monitor quality and safety.
镰刀菌属包含多种真菌，其中有几个物种会在食品中产生霉菌毒素。在本研究中，开发了一种多重聚合酶链反应（PCR）检测方法，用于对产生伏马毒素和单端孢霉烯族毒素的镰刀菌物种进行组特异性检测。用于镰刀菌属水平识别的引物是根据核糖体DNA的内部转录间隔区（ITS1和ITS2）设计的。用于组特异性检测的引物是根据参与单端孢霉烯族毒素生物合成的TRI6基因和参与伏马毒素生物合成的FUM5基因设计的。通过测试与代表14个属的43种真菌的纯化基因组DNA的交叉反应来确定引物特异性，其中包括9种曲霉属、9种镰刀菌属和10种青霉属。以纯化的基因组DNA为模板，每个反应10 pg时可观察到属特异性识别；对于产生单端孢霉烯族毒素的禾谷镰刀菌，每个反应100 pg模板时发生组特异性识别，对于产生伏马毒素的轮枝镰刀菌，每个反应1 ng模板时发生组特异性识别。为了应用该PCR检测方法，开发了一种从玉米粉中分离真菌DNA的方案。在玉米粉中真菌可见生长之前（<10⁵ CFU/g），就完成了禾谷镰刀菌的检测及其与轮枝镰刀菌的区分。这种检测水平与酶联免疫吸附测定等其他方法相当，本文所述的检测方法可用于食品行业监测质量和安全的工作。
