Phylogenetic analysis and rapid identification of Candida dubliniensis based on analysis of ACT1 intron and exon sequences

Abstract

The phylogenetic position of Candida dubliniensis has previously been established on the basis of the sequence of rRNA genes. In order t o confirm the relationship between C. dubhiensis and other yeast species, particularly Candida a/bicans, using non-rRNA gene sequences the ACT1 gene was chosen for analysis. Three overlapping fragments that together span the entire C. dubhiensis ACT1 gene (CdACT1) were amplified from a recombinant phage isolated from a genomic DNA A library using PCR. These were cloned and used t o determine the contiguous sequence of the gene. Analysis of the sequence data revealed the presence of a 1131 bp ORF interrupted by a single 632 bp intron at the 5' extremity of the gene. Comparison of the CdACT1 sequence with the C. albicans homologue (CaACT1) revealed that although the exons are 97-9 O/ O identical the introns are only 83.4% identical. Phylogenetic trees generated using ACT1 exon and intron sequences from a range of yeast species unequivocally confirmed the phylogenetic position of C. dubhiensis as a unique taxon within the genus Candida. Analysis of the ACT1-associated intron sequences from 10 epidemiologically unrelated C. dubhiensis isolates from disparate geographical locations showed a very low level of intraspecies sequence variation. In order t o develop an accurate and rapid method t o identify C. dubliniensis from primary isolation plates the significant divergence between the C. dubhiensis and C. albicans ACT1 intron sequences was exploited by designing C. dubliniensis-specific PCR primers. Using a rapid boiling method to produce template DNA directly from colonies from primary isolation plates in 10 min, these primers were used in a blind test with 122 isolates of C. dubhiensis, 53 isolates of C. albicans, 10 isolates of C. stellatoidea and representative isolates of other clinically relevant Candida and other yeast species. Only the C. dubhiensis isolates yielded the C. dubhiensis-specif ic 288 bp amplimer. Use of this technique on colonies suspected t o be C. dubhiensis allows their correct identification as C. dubhiensis in as little as 4 h.

Typing of Candida albicans Isolates by Sequence Analysis of the Cytochrome b Gene and Differentiation from Candida stellatoidea

“…The opposite occurred when Sullivan et al (23) described Candida dubliniensis, which resembles C. albicans in germ tubes and chlamydospores production, as a new species from the nucleotide differences of the V3 region of the large-subunit rRNA gene sequences of several atypical C. albicans strains. The proposal was supported by Gilfillan et al (6) from the sequence studies of small rRNA genes and was confirmed by Donnelly et al (5) based on the differences of the ACT1 intron and exon sequences of the two yeasts.…”

Occurrence of Candida Species Colonization in a Population of Denture-Wearing Immigrants

“…PCR identification of Candida dublinensis with the Candida dublinensis-specific primer pair DUBF and DUBR (17) was carried out in a 50-Ill final volume containing 10 pmol each of the forward and reverse primers, 2.5 mM MgCI2, 10 mM Tris-HCl (pH 9.0 at 25°C), 10 mM KCl, 0.1% (vol/vol) Triton X-I00, 2.5 U ofTaq DNA polymerase (Promega), and 25 III of template DNA-containing cell supernatant. Cycling conditions consisted of 6 min at 95°C, followed by 30 cycles of 30 s at 94°C, 30 s at 58°C, and 30 s at noc, followed by noc for 10 min.…”

“…Cycling conditions consisted of 6 min at 95°C, followed by 30 cycles of 30 s at 94°C, 30 s at 58°C, and 30 s at noc, followed by noc for 10 min. Candida template DNA for use in PCR experiments with the Candida dubliniensis-specific primer pair DUBF (5'GTATTTGTCGTTCCCCTTTC-3') and DUBR (5'-GTGTTGTGTGCACTAACGTC-3') was prepared as described by Donnelly et al (17).…”

Oral yeast carriage in HIV‐infected and non‐infected populations in Rosario, Argentina

“…When green colonies grew, which is characteristic of both C. albicans and C. dubliniensis, a single colony was selected from each plate. The following tests were performed in order to differentiate both species: growth at 45°C on potato dextrose agar (PDA), 8 D-xylose assimilation, the API 20C Aux system (BioMerieux, Marcy LÕEtoile, France), morphology in sunflower seed agar (SSA) 24 and PCR using primers that hybridise to the class IV intron of the ACT1 gene 25 according to the scheme of identification of Binolfi et al [26]. Colonies of different colours other than green, which correspond to other yeast species, on CHROMagar Candida were isolated and identified using the carbohydrate assimilation and fermentation tests and the API 20C Aux system.…”