Utilization of size polymorphism in ITS1 and ITS2 regions for identification of pathogenic yeast species

Abstract

Purpose. Despite the existence of a variety of available yeast-identification strategies, easier and more cost-effective methods are required for routine use in clinical laboratories. The internal transcribed spacer (ITS) regions of fungal rRNA genes exhibit variable sizes depending on the yeast species. In the present study, fragment size polymorphism (FSP) analysis of the ITS1 and ITS2 regions for identification of the clinically most important yeast species was assessed. Methodology. The ITS1 and ITS2 regions of 190 strains, including isolates of 31 standard strains and 159 clinical isolates, were separately PCR amplified with two primer sets: ITS1-ITS2 and ITS3-ITS4. PCR products were mixed and the two-band electrophoretic pattern of each sample was analysed according to the size of the ITS regions as predicted from the GenBank database. Results. Using this method and avoiding expensive tools such as sequencing or capillary electrophoresis, we were able to differentiate nearly all pathogenic yeast species, including Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei, Candida guilliermondii, Candida kefyr, Candida lusitaniae, Candida rugosa, Cryptococcus neoformans and Saccharomyces cerevisiae. The method showed limited discriminatory power to differentiate species of the Candida parapsilosis complex. Differentiation of Candida albicans and Candida tropicalis needs already identified controls. Conclusion. FSP method benefits from advantages such as lower cost, higher speed and wider range of species than some commercial yeast-identification methods. We consider this method as one of the easiest molecular approaches for identifying a wide range of human pathogenic yeast species, applicable to both diagnostic and epidemiological purposes.