双色实时荧光聚合酶链式反应法检测产玉米赤霉烯酮毒素的黄色镰孢菌

Detection of zearalenone toxin-producing Fusarium culmorum by dual-color real-time fluorescence polymerase chain reaction

中文关键词:  黄色镰孢菌  玉米赤霉烯酮毒素  EF1α基因  PKS基因  双色实时荧光聚合酶链式反应法

英文关键词:Fusarium culmorum  zearalenone toxin  EF1α gene  PKS gene  dual-color real-time fluorescence polymerase chain reaction

中文摘要:

      目的 建立双色实时荧光聚合酶链式反应(polymerase chain reaction, PCR)法同时检测黄色镰孢菌(Fusarium culmorum)及其所产玉米赤霉烯酮毒素合成基因的方法。方法 依据EF1α基因和PKS基因, 优化建立双色实时荧光PCR反应体系和扩增条件; 建立粮食霉变模型, 检测霉变粮食中产毒真菌, 验证方法的特异性、灵敏度和重复性。结果 应用本研究设计的引物和探针, 仅产玉米赤霉烯酮毒素的黄色镰孢菌EF1α和PKS基因出现特异性扩增, 其余真菌扩增结果均为阴性; 检出限达3.5×102 CFU/mL, 重复性良好; 且采用此方法对粮食霉变模型中选取的样品进行检测, 可检出产玉米赤霉烯酮的黄色镰孢菌。结论 该方法可准确鉴定霉变粮食中的产玉米赤霉烯酮毒素的黄色镰孢菌, 实现在真菌毒素未产生前或者产生早期, 对粮食受真菌及真菌毒素污染的情况进行早期监控。

英文摘要:

      Objective To establish a method for simultaneous detection of Fusarium culmorum and its zearalenone toxin synthesis gene by dual-color real-time fluorescence polymerase chain reaction (PCR). Methods According to the EF1α gene and PKS gene, the establishment of a dual-color real-time PCR reaction system and amplification conditions were optimized; the mildew model of grain was established to detect the toxic fungi in the mildew grain, and the specificity, sensitivity and repeatability of the method were verified. Results Using the primers and probes designed in this study, only the EF1α and PKS genes of Fusarium culmorum producing zearalenone toxin were amplified specifically, and the amplification results of other fungi were negative; the limit of detection was 3.5×102 CFU/mL, and the repeatability was good; in addition, the method was adopted for detecting the samples selected in the grain mildew model, and the Fusarium culmorum capable of producing zearalenone could be detected. Conclusion This method can accurately identify Fusarium culmorum producing zearalenone toxin in moldy grain, and realize early monitoring of grain contamination by fungi and mycotoxin before or at the early stage of mycotoxin production.