Aspergillus section Fumigati typing by PCR-restriction fragment polymorphism

Abstract

Recent studies have shown that there are multiple clinically important members of the Aspergillus section Fumigati that are difficult to distinguish on the basis of morphological features (e.g., Aspergillus fumigatus, A. lentulus, and Neosartorya udagawae). Identification of these organisms may be clinically important, as some species vary in their susceptibilities to antifungal agents. In a prior study, we utilized multilocus sequence typing to describe A. lentulus as a species distinct from A. fumigatus. The sequence data show that the gene encoding beta-tubulin, benA, has high interspecies variability at intronic regions but is conserved among isolates of the same species. These data were used to develop a PCR-restriction fragment length polymorphism (PCR-RFLP) method that rapidly and accurately distinguishes A. fumigatus, A. lentulus, and N. udagawae, three major species within the section Fumigati that have previously been implicated in disease. Digestion of the benA amplicon with BccI generated unique banding patterns; the results were validated by screening a collection of clinical strains and by in silico analysis of the benA sequences of Aspergillus spp. deposited in the GenBank database. PCR-RFLP of benA is a simple method for the identification of clinically important, similar morphotypes of Aspergillus spp. within the section Fumigati.

FIG. 1.

PCR-RFLP of benA amplicons. (A) Homology alignment of the benA amplicons from each species with the ClustalW algorithm. Potential BccI sites (5′-CCATCNNNNN-3′) are underlined. Restriction sites centered at A. fumigatus nucleotides 106 and 253 have two site polymorphisms. Species are designated at the left (Af, A. fumigatus; Al, A. lentulus; Nu, N. udagawae). (B) Restriction digest maps of each species. BccI sites are shown at the top of each amplicon.

FIG. 2.

(A) Gel electrophoresis of the fragments generated from the digestion of the benA amplicons with BccI. Lane M, 50-bp DNA ladder (New England Biolabs); lane 1, A. lentulus; lane 2, N. udagawae; lane 3, A. fumigatus; lane 4, N. pseudofischeri; lane 5, N. fischeri; lane 6, A. fumigatus var. ellipticus. The molecular sizes of the DNA standards (in bp) are indicated at the left. (B) Nonspecific (star activity) digestion of BccI when it is used in excess. Gel electrophoresis of the A. fumigatus benA amplicon digested with 10 U of BccI (lanes 1 and 2) or 10 U of XhoI (lane 3) for 1 h at 37°C. Digestion of the benA amplicon with excess XhoI generated the expected 426- and 66-bp DNA fragments. Lane 4, intact A. fumigatus benA amplicon (492 bp); lane M, 50-bp DNA ladder. The molecular sizes of the DNA standards (in bp) are indicated at the left.