Detection of Aspergillus flavus and Aspergillus parasiticus from aflatoxin-contaminated peanuts and their differentiation using PCR-RFLP

The possibility of using PCR to specify the detection of aflatoxigenic fungi in food and feeds was investigated. The method, based on amplification of the aflP gene encoding the biosynthesis of aflatoxins, was optimized for the detection of aflatoxin-producing molds (Aspergillus flavus MTCC 277, A. flavus JH 11 and A. parasiticus MTCC 2796). The specificity of the optimized PCR method was proved with the amplification of genomes from aflatoxin-producing Aspergillus strains. Amplification of DNA from serially diluted spores revealed the detection of A. flavus, A. flavus JH 11 and A. parasiticus with as low as 104 fungal spores mL(-1), whereas spore-contaminated peanuts showed a threshold limit of 108 spores g(-1) at 0 h. Determination of the spore limit from peanut samples was followed by differentiation of aflatoxin-producing A. flavus strains from A. parasiticus by the restriction digestion of the partially amplified aflP gene product. This method allows not only conclusive detection of aflatoxin-producing species, but also simultaneous species differentiation in contaminated agricultural and commercial products.