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

<i>Aspergillus oryzae</i> and <i>Aspergillus flavus</i> are closely related species within the <i>Aspergillus</i> section <i>Flavi</i>, 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 <i>Flavi</i> 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 <i>Flavi</i>, including isolates obtained from <i>nuruk</i>. The analysis revealed that <i>A. oryzae</i> 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 <i>A. oryzae</i> from <i>A. flavus</i> by comparing the sizes of PCR amplicons. Application of these primer sets to <i>nuruk</i>-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 <i>A. oryzae</i> (116) and <i>A. flavus</i> (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 <i>A. oryzae</i> from <i>A. flavus</i>.