Multiplex PCR assay for the detection of aflatoxigenic and non-aflatoxigenic fungi in meju, a Korean fermented soybean food starter

Aflatoxins are toxic secondary metabolites produced commonly by Aspergillus flavus and Aspergillus parasiticus. In this study, the possibility of using multiplex PCR was investigated to speed up and specify the detection of aflatoxigenic Aspergillus species in meju, a traditional Korean fermented soybean food starter. Two different sets of three primers were designed specifically for the omtB, ver-1, aflR, and omtA genes present in the aflatoxin biosynthesis cluster. The optimized multiplex PCR showed that only aflatoxigenic Aspergillus species gave three band patterns in both primer sets. The detection limits were determined as 125 pg/μl for genomic DNA from aflatoxigenic A. parasiticus KCCM 35078, and 10⁵ spores/g of meju sample for DNA extracted directly from meju. A total of 65 Aspergillus isolates from meju were tested for the presence of aflatoxigenic fungi by the application of multiplex PCR, and were analyzed by TLC and HPLC for the aflatoxin production in the culture filtrates. Results showed a good correlation between the presence of the aflatoxin biosynthesis genes analyzed by multiplex PCR and aflatoxin production by TLC and HPLC. This suggests that this multiplex PCR method may provide an accurate and specific detection of aflatoxigenic Aspergillus species in fermented soybean foods.