Identification of Candida dubliniensis, based on ribosomal DNA sequence analysis

Differentiation of Candida albicans and the recently described C. dubliniensis has proven difficult due to the high degree of phenotypic similarity of these species. The present study examines sequence variations in the ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions of C. albicans (n = 5) and C. dubliniensis (n = 7) strains, with a view to identifying sequence differences that would enable consistent differentiation of these two species by restriction fragment length polymorphism (RFLP) analysis. The ITS1 and ITS2 regions, together with the entire 5.8S rRNA gene of the strains, were amplified by the polymerase chain reaction (PCR), using primers ITS1 and ITS4, PCR products from both C. albicans and C. dubliniensis were of similar size (around 540 bp); however, sequence analysis revealed over 20 consistent base differences between the products of the two species. On the basis of sequence variation, the restriction enzyme MspA1 I was selected and used to differentiate the PCR products of C. albicans and C. dubliniensis by RFLP analysis. MspA1 I yielded two discernible fragments from C. albicans PCR products, whilst those from C. dubliniensis appeared undigested, thereby providing an approach to differentiate the two species.