Centre of Molecular and Environmental Biology, Department of Biology, University of Minho, Braga, Portugal.
J Clin Microbiol. 2010 May;48(5):1677-82. doi: 10.1128/JCM.02151-09. Epub 2010 Mar 10.
Among the Candida species causing bloodstream infections, Candida parapsilosis is one of the most frequently isolated. The objective of the present work was the identification of new microsatellite loci able to distinguish among C. parapsilosis isolates. DNA sequences with trinucleotide repeats were selected from the C. parapsilosis genome database. PCR primer sets flanking the microsatellite repeats were designed and tested with 20 independent isolates. On the basis of the amplification efficiency, specificity, and observed polymorphism, four of the sequences were selected for strain typing. Two hundred thirty-three independent C. parapsilosis sensu stricto isolates were genotyped by using these markers. The polymorphic loci exhibited from 20 to 42 alleles and 39 to 92 genotypes. In a multiplex analysis, 192 genotypes were obtained and the combined discriminatory power of the four microsatellites was 0.99. Reproducibility was demonstrated by submission of subcultures of 4 isolates each, in triplicate, interspersed with unique numbers among a group of 30 isolates for blind testing. Comparison of the genotypes obtained by microsatellite analysis and those obtained by randomly amplified polymorphic DNA analysis, restriction fragment length polymorphism analysis, and internal transcribed sequence grouping was performed and showed that the microsatellite method could distinguish individual isolates; none of the other methods could do that. Related species, C. orthopsilosis and C. metapsilosis, were not confused with C. parapsilosis sensu stricto. These new microsatellites are a valuable tool for use for the differentiation of C. parapsilosis sensu stricto strains, vital in epidemiology to answer questions of strain relatedness and determine pathways of transmission.
在引起血流感染的念珠菌属中，近平滑念珠菌是最常分离到的一种。本研究的目的是鉴定新的微卫星标记，以区分近平滑念珠菌的分离株。从近平滑念珠菌基因组数据库中选择具有三核苷酸重复的 DNA 序列。设计了侧翼微卫星重复的 PCR 引物对，并对 20 个独立的分离株进行了测试。基于扩增效率、特异性和观察到的多态性，选择了 4 个序列用于菌株分型。使用这些标记对 233 个独立的近平滑念珠菌进行了基因分型。多态性位点表现出 20 至 42 个等位基因和 39 至 92 个基因型。在多重分析中，获得了 192 个基因型，4 个微卫星的组合鉴别力为 0.99。通过提交每个 4 个的亚培养物，以 30 个分离株为一组进行盲法测试，每个亚培养物重复 3 次，在独特的数字之间穿插进行，证明了可重复性。通过微卫星分析获得的基因型与随机扩增多态性 DNA 分析、限制性片段长度多态性分析和内部转录间隔区分组获得的基因型进行了比较，结果表明微卫星方法能够区分个体分离株；其他方法均无法做到。相关种近平滑念珠菌、副近平滑念珠菌和中间型念珠菌不会与近平滑念珠菌混淆。这些新的微卫星是区分近平滑念珠菌的有效工具，对于流行病学回答菌株相关性问题和确定传播途径至关重要。
