Assessment of ribosomal large-subunit D1-D2, internal transcribed spacer 1, and internal transcribed spacer 2 regions as targets for molecular identification of medically important Aspergillus species

Abstract

Molecular approaches are now being developed to provide a more rapid and objective identification of fungi compared to traditional phenotypic methods. Ribosomal targets, especially the large-subunit RNA gene (D1-D2 region) and internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions), have shown particular promise for the molecular identification of some fungi. We therefore conducted an assessment of these regions for the identification of 13 medically important Aspergillus species: Aspergillus candidus, Aspergillus (Eurotium) chevalieri, Aspergillus (Fennellia) flavipes, Aspergillus flavus, Aspergillus fumigatus, Aspergillus granulosus, Aspergillus (Emericella) nidulans, Aspergillus niger, Aspergillus restrictus, Aspergillus sydowii, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor. The length of ribosomal regions could not be reliably used to differentiate among all Aspergillus species examined. DNA alignment and pairwise nucleotide comparisons demonstrated 91.9 to 99.6% interspecies sequence identities in the D1-D2 region, 57.4 to 98.1% in the ITS1 region, and 75.6 to 98.3% in the ITS2 region. Comparative analysis using GenBank reference data showed that 10 of the 13 species examined exhibited a < or = 1-nucleotide divergence in the D1-D2 region from closely related but different species. In contrast, only 5 of the species examined exhibited a < or = 1-nucleotide divergence from sibling species in their ITS1 or ITS2 sequences. Although the GenBank database currently lacks ITS sequence entries for some species, and major improvement in the quality and accuracy of GenBank entries is needed, current identification of medically important Aspergillus species using GenBank reference data seems more reliable using ITS query sequences than D1-D2 sequences, especially for the identification of closely related species.

FIG. 1.

Alignment of complete Aspergillus D1-D2 regions illustrating the sequence divergence among medically important species. The highly conserved region of the 28S rRNA gene intervening between the D1 and D2 regions has been omitted. Dots indicate identical nucleotides compared to the leader sequence (A. fumigatus strain ATCC 1022); dashes indicate alignment gaps. Sequence data were derived from this study (for GenBank accession numbers, see Table 1).

FIG. 2.

Alignment of complete Aspergillus ITS1-ITS2 regions illustrating the sequence divergence among medically important species. The highly conserved intervening 5.8S rRNA gene has been omitted. Dots symbolize identical nucleotides compared to the leader sequence (A. fumigatus strain ATCC 1022); dashes indicate alignment gaps. Sequence data were derived from this study (for GenBank accession numbers, see Table 1).