产毒真菌多重实时荧光PCR快速检测方法建立

Establishment of rapid detection method for multiple real-time fluorescent PCR of toxin-producing fungi

目的建立一种能够在单体系中同时检测产黄曲霉毒素真菌及3种潜藏性产毒真菌的多重实时荧光PCR快速检测方法。方法分别根据产黄曲霉毒素真菌、赭曲霉、青霉和镰刀菌的蛋白活化基因aflR、ITS序列、β-微管蛋白编码区、LS rRNA,设计引物和探针,通过优化反应组分和反应条件,建立了可同时检测常见产毒真菌的实时荧光PCR方法,并对该方法的灵敏度和特异性进行了分析。结果优化建立的反应体系对产黄曲霉毒素真菌、赭曲霉、青霉和镰刀菌的检测限分别可达到3.37×10^4 、1.91×10^4 、1.53×10^4 和3.95×10^4 拷贝/反应。结论本研究建立的多重实时荧光PCR检测方法特异性强,灵敏度高,可在2 h内完成对产毒真菌的检测。

OBJECTIVE To establish a multiplex real-time fluorescent PCR rapid detection method for simultaneous detection of aflatoxin-producing fungi and three latent toxin-producing fungi in a single system.METHODS Primers and probes were designed based on the protein activation genes AflR,ITS sequence,β-tubulin coding region and LS rRNA of aflatoxin-producing fungi,Aspergillusochraceus,Penicillium and Fusarium,respectively.A real-time fluorescent PCR method for simultaneous detection of commontoxin-producing fungi was performed,and the sensitivity and specificity of the method were analyzed by optimizing the reaction components and reaction conditions.RESULTS The detection limits of the optimized reaction system for aflatoxin-producing fungi,Aspergillusochraceus,Penicillium and Fusarium were 3.37×104,1.91×104,1.53×104,3.95×10^4 copies/reaction,respectively.CONCLUSION The multiplex real-time fluorescent PCR assay established in this study is highly specific and sensitive.It can detect toxin-producing fungi within 2 hours and provide technical support for fungal monitoring in food.