Microsatellite markers for typing Aspergillus fumigatus isolates

Abstract

The use of microsatellites as highly polymorphic DNA markers for the typing of isolates of Aspergillus fumigatus was investigated. Four CA repeats were selected by screening an A. fumigatus DNA library with a (CA)10 oligonucleotide. Primers flanking these CA repeats were designed to amplify each locus. One primer of each pair was labeled with a fluorophore, and the PCR products were analyzed with an automatic sequencer and the GeneScan software. For each primer set and for a given isolate, one band was detected and was assigned to an allele because A. fumigatus is haploid. With 50 clinical isolates, 50 environmental isolates, and 2 reference strains we obtained 12, 11, 10, and 23 different alleles for the four CA microsatellites, respectively (discriminatory power, 0.994). The results were identical by whatever DNA extraction technique was used. Interestingly, no clustering between environmental and clinical isolates was observed, suggesting that every isolate is potentially pathogenic. Microsatellite markers appear suitable for use in large epidemiological studies of invasive aspergillosis.

FIG. 1

GeneScan analysis of PCR profiles obtained with two independent isolates (isolates A560 and A422). PCRs were performed with a 6-FAM-labeled primer (loci A, B, and C) or a HEX-labeled primer (locus D), and the PCR products for each microsatellite locus (A, B, C, and D) were run in an acrylamide-urea gel. Bands produced fluorescent peaks, and their molecular sizes were automatically determined by comparison to the TAMRA-labeled GeneScan internal size standards loaded in each well (data not shown). The numbers refer to the sizes (in base pairs) of the PCR products by considering the fluorescent peak with the maximum height.

FIG. 2

Allele size distributions of A. fumigatus isolates at microsatellite loci A (A), B (B), C (C), and D (D) upon analysis of 50 environmental isolates (solid bars) and 52 clinical isolates including 2 reference strains (striped bars).

FIG. 3

Correspondence analysis performed for all alleles showing the dispersion observed for the 102 A. fumigatus isolates. The projection in the plane defined by the two most informative axes is shown. Squares, environmental isolates; circles, clinical isolates; empty symbols, unique profiles; shaded symbols, profiles shared by two or more isolates. The two ellipses contain 90% of the individuals of each subgroup (dotted line for patient isolates; continuous line for environmental isolates). The centers of the ellipses are indicated, with a black circle for the patient isolate ellipse and a black square for the environmental isolate ellipse.