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

Abstract

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. 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. The detection limits of the optimized reaction system for aflatoxin-producing fungi, Aspergillusochraceus, Penicillium and Fusarium were 3. 37×10~4, 1. 91×10~4, 1. 53×10~4, 3. 95×10~4 copies/reaction, respectively. 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.