Donnelly Samantha M, Sullivan Derek J, Shanley Diarmuid B, Coleman David C
Microbiology (Reading). 1999 Aug;145 ( Pt 8):1871-1882. doi: 10.1099/13500872-145-8-1871.
The phylogenetic position of Candida dubliniensis has previously been established 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 for analysis. Three overlapping fragments that together span the entire C. dubliniensis ACT1 gene (CdACT1) were amplified from a recombinant phage isolated 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 with the C. albicans homologue (CaACT1) revealed that although the exons are 97.9% 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. dubliniensis as a unique taxon within the genus Candida. Analysis of the ACT1-associated intron sequences from 10 epidemiologically unrelated C. dubliniensis isolates from disparate geographical locations showed a very low level of intraspecies sequence variation. In order to develop an accurate and rapid method to identify C. dubliniensis from primary isolation plates the significant divergence 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 122 isolates of C. dubliniensis, 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. dubliniensis isolates yielded the C. dubliniensis-specific 288 bp amplimer. Use of this technique on colonies suspected to be C. dubliniensis allows their correct identification as C. dubliniensis in as little as 4 h.
都柏林念珠菌的系统发育位置此前已根据rRNA基因序列确定。为了利用非rRNA基因序列证实都柏林念珠菌与其他酵母物种，特别是白色念珠菌之间的关系，选择ACT1基因进行分析。使用PCR从从基因组DNA λ文库分离的重组噬菌体中扩增出三个重叠片段，它们共同覆盖了整个都柏林念珠菌ACT1基因（CdACT1）。将这些片段克隆并用于确定该基因的连续序列。序列数据分析显示存在一个1131 bp的开放阅读框，该开放阅读框在基因的5'末端被一个632 bp的单一内含子中断。将CdACT1序列与白色念珠菌同源物（CaACT1）进行比较，结果显示，尽管外显子的同源性为97.9%，但内含子的同源性仅为83.4%。使用一系列酵母物种的ACT1外显子和内含子序列生成的系统发育树明确证实了都柏林念珠菌作为念珠菌属内一个独特分类单元的系统发育位置。对来自不同地理位置的10株流行病学无关的都柏林念珠菌分离株的ACT1相关内含子序列进行分析，结果显示种内序列变异水平非常低。为了开发一种从原始分离平板中准确快速鉴定都柏林念珠菌的方法，通过设计都柏林念珠菌特异性PCR引物，利用都柏林念珠菌和白色念珠菌ACT1内含子序列之间的显著差异。使用快速煮沸法在10分钟内直接从原始分离平板上的菌落中制备模板DNA，这些引物用于对122株都柏林念珠菌、53株白色念珠菌、10株星状念珠菌以及其他临床相关念珠菌和其他酵母物种的代表性分离株进行盲测。只有都柏林念珠菌分离株产生了都柏林念珠菌特异性的288 bp扩增产物。将该技术应用于疑似都柏林念珠菌的菌落，可在短短4小时内将其正确鉴定为都柏林念珠菌。
