分子系統樹解析とマルチプレックスPCRを用いたAspergillus flavusグループの識別

摘要

We describe a simple method for discrimination of Aspergillus flavus group fungi, including aflatoxin (AF) producers, by means of molecular-biological analysis of 45 strains of A. flavus. First, 20 strains of A. flavus were compared using phylogenetic tree analysis based on the nucleotide sequences of ITS 1-5.8S-ITS 2 (ITS-1/2) and aflR-aflJ intergenic spacer (aflR/J-IGS), In this analysis, the tested strains were discriminated into 4 groups at the aflR/J-IGS region. Although ITS-1/2 region analysis could not discriminate between A. flavus (AF producers) and A. oryzae/A. flavus (AF nonproducers), aflR/J-IGS region analysis could discriminate between these groups. Moreover, 45 strains of A. flavus were compared by means of both phylogenetic tree analysis based on the aflR/J-IGS region and the conventional afiatoxin production test (culture method). The phylogenetic tree analysis of the tested strains was consistent with the findings of the culture method. In addition, 49 strains of A. flavus and related species (Aspergillus spp.) were tested by multiplex PCR with primers designed on the basis of the phylogenetic tree analysis. These results were consistent with phylogenetic tree analysis based on the aflR/J-IGS region for 41 strains.%アフラトキシン(AF)産生菌を含むAspergillus flavusグループの簡易な識別方法を構築するために，A. flavusグループ45株について分子生物学的な解析を行った.最初に，A. flavusグループ20株についてITS 1-5.8S-ITS 2(ITS-1/2)領域とaflR-aflJ intergenic spacer (aflR/J-IGS)領域の分子系統樹を比較した.その結果，aflR/J-IGS領域に基づいた解析ではITS-1/2領域の解析で識別できなかったA. flavus(AF産生株)とA.oryzae/A. flavus(AF非産生株)の識別が可能であった.また，A.flavusグループ45株を用いてaflR/J-IGS領域の分子系統樹解析と培養法によるアフラトキシン産生性を比較した.その結果，aflR/J-IGS領域の分子系統樹解析で推定されたアフラトキシン産生性と培養法で得られた結果が一致した.これら分子系統樹解析の結果に基づき設計したプライマーを用いてA.flavusを中心とした49株のAspergillus属菌を対象にマルチプレックPCR法による識別を試みた.その結果，供試41株でaflR/J-IGSの分子系統樹解祈と同様の結果が得られた.