Use of a novel panel of nine short tandem repeats for exact and high-resolution fingerprinting of Aspergillus fumigatus isolates - PubMed (original) (raw)

Use of a novel panel of nine short tandem repeats for exact and high-resolution fingerprinting of Aspergillus fumigatus isolates

Hanneke A de Valk et al. J Clin Microbiol. 2005 Aug.

Abstract

Here we describe a new panel of short tandem repeats (STRs) for a novel exact typing assay that can be used to discriminate between Aspergillus fumigatus isolates. A total of nine STR markers were selected from available genomic A. fumigatus sequences and were divided into three multicolor multiplex PCRs. Each multiplex reaction amplified three di-, tri-, or tetranucleotide repeats, respectively. All nine STR markers were used to analyze 100 presumably unrelated A. fumigatus isolates. For each marker, between 11 and 37 alleles were found in this population. One isolate proved to be a mixture of at least two different isolates. With the remaining 99 isolates, 96 different fingerprinting profiles were obtained. The Simpson's diversity index for the individual markers ranged from 0.77 to 0.97. The diversity index for the multiplex combination of di-, tri-, and tetranucleotide repeats ranged from 0.9784 to 0.9968. The combination of all nine markers yielded a Simpson's diversity index of 0.9994, indicative of the high discriminatory power of these new loci. In theory, this panel of markers is able to discriminate between no less than 27 x 10(9) different genotypes. The multicolor multiplex approach allows large numbers of markers to be tested in a short period of time. The exact nature of the assay combines high reproducibility with the easy exchange of results and makes it a very suitable tool for large-scale epidemiological studies.

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Figures

FIG.1.

FIG.1.

Examples of amplification results. The principle peak used for analysis is indicated with a colored dot. Blue traces represent the FAM label, black traces represent the HEX label, and green traces represent the TET label. Typical results obtained with the dinucleotide repeats (A), trinucleotide repeats (B), and tetranucleotide repeats (C) are shown. (D) Typical example of amplification artifacts obtained with a dinucleotide repeat. The principle peak is indicated with an “n.” A second peak exactly 1 bp shorter is the result of incomplete A addition (hence, n − a). A stutter peak one repeat shorter is indicated with n − 1. Other artifact peaks are indicated accordingly. (E) Multiple stutter peaks with a trinucleotide repeat do not influence the ability to identify the principle peak in a sample. (F) A typical result obtained with a mixed sample.

FIG. 2.

FIG. 2.

Determination of repeat numbers by DNA sequencing. Examples are shown for a dinucleotide repeat (A), trinucleotide repeat (B), and tetranucleotide repeat (C).

FIG. 3.

FIG. 3.

Dendrogram based on profiles of nine STR_Af_ markers from 99 presumably unrelated A. fumigatus isolates. The dendrogram was created by using BioNumerics, version 3.5, software (Applied Maths) and the unweighted pair group method with arithmetic averages method with the multistate categorical similarity coefficient. See the text for further details.

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