The study aimed to develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of three major mycotoxin metabolic pathway genes, namely tri8 (T-2 toxin), tri6 (trichothecene) and pks4 (zearalenone), along with competitive internal amplification control. Specific primers for each of the aforementioned genes were optimized and validated using 14 reference strains and 10 pure culture isolates. The optimized mPCR assay detected the three metabolic pathway genes in artificially contaminated maize samples with a sensitivity of 2 × 10³ CFU per g for tri6 and pks4 positive Fusarium strains, whereas 2 × 10⁴ CFU per g for tri8 positive Fusarium strains. Application of the developed mPCR assay to 30 cereal and 20 feed samples revealed 24% (12 of 50) contamination with either one or more mycotoxins. The results of mPCR assay were further evaluated with high performance liquid chromatography (HPLC), and both methods provided unequivocal results. This mPCR assay might be a supplementary tool to conventional mycotoxin analytical techniques like thin-layer chromatography, HPLC, etc. The current mPCR assay is a rapid and reliable tool for simultaneous, sensitive and specific detection of T-2, zearalenone and trichothecene producing Fusarium spp. from naturally contaminated foods and to monitor them during the processing steps of food and feed commodities.

中文翻译：

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用于检测产 T-2 和玉米赤霉烯酮的镰刀菌的多重 PCR 的开发和评价

该研究旨在开发和评估多重聚合酶链反应测定 (mPCR)，用于同时检测三种主要的霉菌毒素代谢途径基因，即tri8（T-2 毒素）、tri6（单端孢霉烯）和pks4（玉米赤霉烯酮），以及竞争性内部放大控制。使用 14 个参考菌株和 10 个纯培养分离株对上述每个基因的特异性引物进行了优化和验证。优化的 mPCR 分析检测了人工污染的玉米样品中的三种代谢途径基因， 对于tri6和pks4阳性镰刀菌菌株的灵敏度为 2 × 10 ³ CFU/g ，而 2 × 10 ⁴tri8阳性镰刀菌菌株的 CFU 每克。将开发的 mPCR 检测应用于 30 个谷物和 20 个饲料样品显示 24%（50 个中的 12 个）被一种或多种真菌毒素污染。用高效液相色谱 (HPLC) 进一步评估了 mPCR 测定的结果，两种方法都提供了明确的结果。这种 mPCR 检测可能是传统真菌毒素分析技术（如薄层色谱、HPLC 等）的补充工具。当前的 mPCR 检测是同时、灵敏和特异性检测 T-2、玉米赤霉烯酮和单端孢霉烯镰刀菌的快速可靠工具属 从自然污染的食品中提取，并在食品和饲料商品的加工步骤中对其进行监测。