Identification of clinical mold isolates by sequence analysis of the internal transcribed spacer region, ribosomal large-subunit D1/D2, and β-tubulin

Abstract

Background: The identification of molds in clinical laboratories is largely on the basis of phenotypic criteria, the classification of which can be subjective. Recently, molecular methods have been introduced for identification of pathogenic molds in clinical settings. Here, we employed comparative sequence analysis to identify molds.

Methods: A total of 47 clinical mold isolates were used in this study, including Aspergillus and Trichophyton. All isolates were identified by phenotypic properties, such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing, targeting the internal transcribed spacer (ITS) region, the D1/D2 region of the 28S subunit, and the β-tubulin gene, were performed using primers described previously. Comparative sequence analysis by using the GenBank database was performed with the basic local alignment search tool (BLAST) algorithm.

Results: For Aspergillus, 56% and 67% of the isolates were identified to the species level by using ITS and β-tubulin analysis, respectively. Only D1/D2 analysis was useful for Trichophyton identification, with 100% of isolates being identified to the species level. Performances of ITS and D1/D2 analyses were comparable for species-level identification of molds other than Aspergillus and Trichophyton. In contrast, the efficacy of β-tubulin analysis was limited to genus identification because of the paucity of database information for this gene.

Conclusions: The molecular methods employed in this study were valuable for mold identification, although the different loci used had variable usefulness, according to mold genus. Thus, a tailored approach is recommended when selecting amplification targets for molecular identification of molds.

Keywords: D1/D2; Internal transcribed spacer; Molds; Sequencing; Tubulin.