Differentiation of Candida albicans and Candida dubliniensis by fluorescent in situ hybridization with peptide nucleic acid probes

The recent discovery of Candida dubliniensis as a separate species that tra ditionally has been identified as Candida albicans has led to the developme nt of a variety of biochemical and molecular methods for the differentiatio n of these two pathogenic yeasts. rRNA sequences are well-established phylo genetic markers, and probes targeting species-specific rRNA sequences have been used in diagnostic assays for the detection and identification of micr oorganisms. Peptide nucleic acid (PNA) is a DNA mimic with improved hybridi zation characteristics, and the neutral backbone of PNA probes offers signi ficant advantages in whole-cell in situ hybridization assays. In this study , we developed PNA probes targeting the rRNAs of C. albicans and C. dublini ensis and applied them to a fluorescence in situ hybridization method (PNA FISH) for differentiation between C. albicans and C. dubliniensis. Liquid c ultures were smeared onto microscope slides, heat fixed, and then hybridize d for 30 min. Unhybridized PNA probe was removed by washing, and smears wer e examined by fluorescence microscopy. Evaluation of the PNA FISH method us ing smears of 79 C. dubliniensis and 70 C. albicans strains showed 100% sen sitivity and 100% specificity for both PNA probes. We concluded that PNA FI SH is a powerful tool for the differentiation of C. albicans and C. dublini ensis.