Comparative sequencing of ITS and D1/D2 domains of ribosomal DNA for Molecular discrimination of fungal moulds.


Objectives: To compare and validate the Internal Transcribed Spacer (ITS) and D1/D2 domain of the large ribosomal subunit as potential targets for discrimination of fungal species.

Methods: A total of 80 fungal species previously isolated from clinical specimens collected from patients with Cancer and pulmonary tuberculosis were included in this study. All the fungal isolates were presumptively identified based on their macro- and micro-morphological (phenotypic) characteristics. The fungal DNA was amplified targeting the ITS and D1/D2 domains of LSU using ITS1/ITS4 and D1/D2 primers followed by sequencing the amplicons and compared with the curated molecular database for species confirmation. 

Results: All the 80 isolates were preliminarily identified and speciated based on phenotypic characteristics. The ITS and D1/D2 domains of the ribosomal DNA were successfully amplified for species discrimination using fungus-specific universal primer ITS1/ITS4 and D1/D2 respectively. The species were confirmed as Aspergillus (A. fumigatus, A. flavus, A. niger, A. terrus, A. nidulans, A. ocharceus, A. versicolor) Mucormycetes (Rhizopus, Mucor, Rhizomucor, Syncephalastrum and Cunninghamella), Penicillium (P. oxalicum, P. chrysogenum and P. purpurogenum), Hyalohyphomycetes (Fusarium, Acremonium and Paecilomyces), Dermatophytes (Trichophyton, Microsporum), Phaeohyphomycetes (Alternaria, Curvularia and Bipolaris) and yeast like fungi (Trichosporon, Geotrichum and Rhodotorula). The primer targeting ITS region was found to be more reliable in discriminating the species of Aspergillus, Penicillium and Dermatophytes than D1/D2 primer.  The sequencing results were also compared with phenotypic identifications. 

Conclusion: Sequencing of the ribosomal genes has emerged as a useful diagnostic tool for the rapid detection and identification of pathogenic fungi. In the present study, the utility of the ITS and D1/D2 domain of ribosomal DNA as amplification targets for sequence analysis of fungal species was compared. Both the domains yielded comparable performances for fungal species discrimination, while ITS region was the most reliable primary sequencing target for discrimination of majority of the species under different genera. Analysis of the D1/D2 region could be used for further resolution, since the relevance of species-level identification has only been determined for a limited number of fungal genera.

6 / Comparative sequencing of ITS and D1/D2 domains of ribosomal DNA for Molecular discrimination of fungal moulds