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

The phylogenetic position of Candida dubliniensis has previously been estab lished on the basis of the sequence of rRNA genes. In order to confirm the relationship between C. dubliniensis and other yeast species, particularly Candida albicans, using non-rRNA gene sequences the ACT1 gene was chosen fo r analysis. Three overlapping fragments that together span the entire C dub liniensis ACT1 gene (CdACT1) were amplified from a recombinant phage isolat ed from a genomic DNA lambda library using PCR. These were cloned and used to 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 w ith the C. albicans homologue (CaACT1) revealed that although the exons are 97.9% identical the introns are only 83.4% identical. Phylogenetic trees g enerated using ACT1 exon and intron sequences from a range of yeast species unequivocally confirmed the phylogenetic position of C. dubliniensis as a unique taxon within the genus Candida. Analysis of the ACT1-associated intr on sequences from 10 epidemiologically unrelated C. dubliniensis isolates f rom disparate geographical locations showed a very low level of intraspecie s sequence variation. In order to develop an accurate and rapid method to i dentify C. dubliniensis from primary isolation plates the significant diver gence between the C. dubliniensis 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 1 22 isolates of C. dubliniensis, 53 isolates of C. albicans, 10 isolates of C. stellatoidea and representative isolates of other clinically relevant Ca ndida and other yeast species. Only the C. dubliniensis isolates yielded th e C. dubliniensis-specific 288 bp amplimer. Use of this technique on coloni es suspected to be C. dubliniensis allows their correct identification as C . dubliniensis in as little as 4 h.