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

## TL;DR

This study develops a rapid PCR method to distinguish between Aspergillus oryzae and aflatoxin-producing Aspergillus flavus based on differences in their aflatoxin gene clusters.

## Contribution

A novel PCR-based method using unique deletion patterns in the aflatoxin biosynthesis gene cluster to differentiate A. oryzae from A. flavus.

## Key findings

- A. oryzae shows distinct deletions in the aflatoxin biosynthesis gene cluster compared to A. flavus.
- Four primer sets were designed to differentiate the species with 92% accuracy using PCR amplicon size.
- In silico PCR validated the method's effectiveness across 116 A. oryzae and 482 A. flavus genomes.

## Abstract

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.