Distinction Between Aspergillus oryzae and Aflatoxigenic Aspergillus flavus by Rapid PCR Method Based on the Comparative Sequence Analysis of the Aflatoxin Biosynthesis Gene Cluster

초록

Aspergillus oryzae and Aspergillus flavus are closely related species within the Aspergillus section Flavi, sharing approximately 99.5% genomic similarity. Despite this similarity, they differ markedly in their ability to produce aflatoxin, a carcinogenic mycotoxin synthesized by the aflatoxin biosynthesis gene cluster (ABGC). Species and strains included within section Flavi display diverse deletion patterns in the ABGC at the sequence level. In this study, we performed an in-depth comparative analysis of the ABGC of 30 strains belonging to section Flavi, including isolates obtained from nuruk. The analysis revealed that A. oryzae exhibits distinct large-scale or locus-specific deletions in the ABGC compared to other related species. Based on these unique deletion patterns, we designed four primer sets to distinguish A. oryzae from A. flavus by comparing the sizes of PCR amplicons. Application of these primer sets to nuruk-derived isolates enabled successful species differentiation with 92% accuracy. To further validate this method, in silico PCR analysis was conducted using publicly available genomes of A. oryzae (116) and A. flavus (482), confirming that the developed biomarkers could consistently distinguish between the two close species. The primer sets are expected to serve as a rapid, accurate, and practical method for distinguishing A. oryzae from A. flavus.

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