2006 Volume 56 Issue 2 Pages 77-84
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 Aspergillus section Flavi species. The HPA method involves formation of heteroduplexes with a set of reference fragments of the ITS regions amplified from A. flavus, A. parasiticus, A. tamarii and A. nomius, 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 Aspergillus section Flavi species. Based on the present results, 19 HPA types were identified among a total of 351 section Flavi isolates: F-1 to F-6 of A. flavus/A. oryzae, P-1 and P-2 of A. parasiticus/A. sojae, T-1 of A. tamarii, T-2 of A. pseudotamarii, T-3 of A. caelatus, N-1 to N-5 of A. nomius, TN-1 and TN-2 of A. bombysis, and FP-1 of a new genotype that is potentially aflatoxigenic. Direct identification of section Flavi 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.
