IDENTIFICATION OF PATHOGENIC CANDIDA SPECIES: PCR-FRAGMENT SIZE POLYMORPHISM (PCR-FSP) METHOD

Background: The clinical importance of yeast infections has increased in recent decades. There are 10-15 pathogenic C andida species. The current morphological and physiological methods for identification of C andida species are generally not easy to interpret and may be expensive or time-consuming. In the present study, we introduce and use a new approach for the identification and differentiation of medically important yeast species of C andida . In this method, size polymorphism of the internal transcribed spacer regions, ITS1 and ITS2 , of the ribosomal DNA in various C andida species is used as the basis of species recognition. Methods: The genomic DNA of 31 standard strains and 60 clinical isolates was extracted and PCR -amplified using two primer pairs ( ITS1-ITS2 and ITS3-ITS4 ) separately. Both PCR products were mixed and analyzed after standard agarose gel electrophoresis. The species of the tested yeasts were identified by the electrophoretic patterns of the mixed PCR products of each sample, comparing the data obtained from the sequence analyses of ITS1 and ITS2 molecules. Results: By this method, with the exception of C . albicans and C . dubliniensis , we were able to clearly differentiate nearly all common pathogenic C andida species, including C . albicans, C . glabrata, C . gulliermondii, C . parapsilosis, C . tropicalis, C . krusei, C . kefyr, C . lusinaniae and C . rugosa. All standard and clinical strains were identified correctly, without expensive methods such as sequencing and capillary electrophoresis. Conclusion: It seems that the PCR-FSP method introduced in this study is the easiest molecular approach for the identification of a wide range of pathogenic C andida species and is applicable for diagnostic and epidemiological purposes in reference laboratories. Abstract