分子生物学的手法を用いたカビ同定法について Fungal identification with molecular biological techniques : Heteroduplex panel analysis for identification of Aspergillus section Flavi species

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Abstract

近年,カビの同定にも分子生物学手法が使用されるようになってきた.標的遺伝子として分類・同定に最もよく使用されているのがリボゾームRNA遺伝子(rDNA)である.本稿では筆者らが考案した迅速簡便な<i>Aspergillus</i> section <i>Flavi</i> の種の同定法であるHeteroduplex Panel Analysis(HPA)法を中心に紹介する.また,最近,苦情食品等から分離したカビを同定する方法として,BLASTプログラムによるrDNAのホモロジー検索が利用されるようになってきた.これは16S rDNAの情報により分類体系が再構築されている細菌でよく用いられている同定法である.本稿では、分子生物学的手法でカビを同定する場合の利点と限界点についても考察する.

Recent advances in molecular technology have allowed the rapid detection and identification of filamentous fungi. Ribosomal DNA (rDNA) has been utilized most frequently for phylogenetic studies because it is present and highly conserved in all organisms. This report described a new PCR-based method for heteroduplex panel analysis (HPA) for identification of <i>Aspergillus</i> section <i>Flavi</i> species. The HPA method involves formation of heteroduplexes with a set of reference fragments of the ITS regions amplified from <i>A. flavus</i>, <i>A. parasiticus</i>, <i>A. tamarii</i> and <i>A. nomius</i>, and subsequent minislab hydrolink-mutation detection enhancement (MDE) gel electrophoresis. The test panel is compared with species-specific standard panels (F-1, P-1, T-1 and N-1) generated by pairwise reannealing among four reference fragments. DNA sequencing revealed that the HPA method has the highest sensitivity for detecting single-nucleotide diversity within <i>Aspergillus</i> section <i>Flavi</i> species. Based on the present results, 19 HPA types were identified among a total of 351 section <i>Flavi</i> isolates: F-1 to F-6 of <i>A. flavus</i>/<i>A. oryzae</i>, P-1 and P-2 of <i>A. parasiticus</i>/<i>A. sojae</i>, T-1 of <i>A. tamarii</i>, T-2 of <i>A. pseudotamarii</i>, T-3 of <i>A. caelatus</i>, N-1 to N-5 of <i>A. nomius</i>, TN-1 and TN-2 of <i>A. bombysis</i>, and FP-1 of a new genotype that is potentially aflatoxigenic. Direct identification of section <i>Flavi</i> isolates can be accomplished by HPA without morphological observation. Recently, basic local alignment search tool (BLAST) analysis using rDNA has tentatively been employed for identification of filamentous fungi isolated from food and environmental sources. The advantages and the critical points of the method are also described.

Journal

  • JSM Mycotoxins

    JSM Mycotoxins 56(2), 77-84, 2006-07-31

    Japanese Society of Mycotoxicology

References:  29

Codes

  • NII Article ID (NAID)
    10021919897
  • NII NACSIS-CAT ID (NCID)
    AN00334513
  • Text Lang
    ENG
  • Article Type
    REV
  • ISSN
    02851466
  • Data Source
    CJP  J-STAGE 
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