Heteroduplex panel analysis, a novel method for genetic identification of Aspergillus Section Flavi strains

For genetic identification of Aspergillus Section Flavi isolates and detection of intraspecific variation, we developed a novel method for heteroduplex panel analysis (HPA) utilizing fragments of the internal transcribed spacer (ITS) regions (ITS1-5.8S-ITS2) of the rRNA gene that was PCR amplified with universal primers. The method involves formation of heteroduplexes with a set of reference fragments amplified from Aspergillus flavus, A. parasiticus, A. tamarii, and A. nomius and subsequent minislab vinyl polymer 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. Of 90 test panels, 89 succeeded in identifying the species and 74 were identical to one of the four standard panels. Of the 16 new panels, 11 A. flavus/A. oryzae panels were identical and typed as F-2 and 4 of 5 A. nomius panels were typed as N-2 or N-3. The other strain, A. nomius IMI 358749, was unable to identify the species because no single bands were formed with any of the four reference strains. DNA sequencing revealed that our HPA method has the highest sensitivity available and is able to detect as little as one nucleotide of diversity within the species. When Penicillium or non-Section Flavi Aspergillus was subjected to HPA, the resulting bands of heteroduplexes showed apparently lower mobility and poor heteroduplex formation. This indicates that HPA is a useful identification method without morphological observation and is suitable for rapid and inexpensive screening of large numbers of isolates. The HPA typing coincided with the taxonomy of Section Flavi and is therefore applicable as an alternative to the conventional methods (Samson, R. A., E. S. Hoekstra, J. C. Frisvad, and O. Filtenborg, p. 64-97, in Introduction to Food- and Airborne Fungi, 6th ed., 2000).