Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, PR China.
Med Mycol. 2013 Apr;51(3):300-8. doi: 10.3109/13693786.2012.723223. Epub 2012 Nov 28.
We developed a reverse line blot (RLB) hybridization-, and rolling circle amplification (RCA)-based assays for the identification of Trichoporon species and evaluated them with 48 isolates that had been previously recognized as belonging to eight species (Trichosporon asahii, T. cutaneum, T. dermatis, T. domesticum, T. inkin, T. japonicum, T. jirovecii, and T. laibachii). Results were compared to those obtained with DNA sequencing of three rRNA gene loci, i.e., the internal transcribed spacer (ITS) region, D1/D2 domain of the 28S rRNA gene and intergenic spacer 1 (IGS1) region. Using species-specific, or group-specific probes targeted at the ITS region and the D1/D2 domain, the RLB assay permitted accurate species identification of all 48 isolates with 100% specificity. Species-specific RLB probes correctly assigned 45/48 (94%) of the isolates (six species) with the exception of T. dermatis and T. japonicum isolates which were not targeted by the assay. Identification of T. dermatis relied on a positive hybridization result with the group-specific probe hybridizing with T. dermatis and T. jirovecii and the absence of a signal with the T. jirovecii-specific probe. T. japonicum strains were first assigned to the T. asahii-T. japonicum group by hybridization with the two species group-specific probe and then as T. japonicum by the absence of signal with a T. asahii-specific probe. Twelve species-specific RCA probes targeting the eight species studied detected templates of all 48 Trichosporon isolates and an artificial template of T. asteroides, all with good specificity. Both RLB and RCA are potential alternatives to DNA sequencing for the identification of Trichosporon species. The RLB approach is suited for the batched simultaneous analysis of large numbers of isolates, while RCA is more appropriate for the immediate study of single isolates. Comparative costs are US$7 and US$2 per assay for the RLB and RCA methods, respectively.
我们开发了一种基于反向线杂交（RLB）和滚环扩增（RCA）的方法，用于鉴定 Trichoporon 种，并使用 48 株先前被鉴定为属于八个种（Trichosporon asahii、T. cutaneum、T. dermatis、T. domesticum、T. inkin、T. japonicum、T. jirovecii 和 T. laibachii）的分离株进行了评估。结果与通过三个 rRNA 基因座（即内部转录间隔区（ITS）区域、28S rRNA 基因的 D1/D2 结构域和基因间间隔 1（IGS1）区域）的 DNA 测序获得的结果进行了比较。使用针对 ITS 区域和 D1/D2 结构域的种特异性或组特异性探针，RLB 检测法可以 100%特异性准确鉴定所有 48 株分离株。种特异性 RLB 探针正确鉴定了 45/48（94%）株（六个种），除了该检测法未针对的 T. dermatis 和 T. japonicum 分离株。T. dermatis 的鉴定依赖于与 T. dermatis 和 T. jirovecii 杂交的组特异性探针的阳性杂交结果，以及与 T. jirovecii 特异性探针无信号。T. japonicum 株首先通过与两个种特异性探针杂交，分配到 T. asahii-T. japonicum 组，然后通过与 T. asahii 特异性探针无信号，鉴定为 T. japonicum。针对所研究的八个种的 12 种特异性 RCA 探针检测到了所有 48 株 Trichosporon 分离株的模板，以及一个 T. asteroides 的人工模板，均具有良好的特异性。RLB 和 RCA 均为鉴定 Trichosporon 种的 DNA 测序的潜在替代方法。RLB 方法适合于同时分析大量分离株的批量分析，而 RCA 更适合于单个分离株的即时研究。RLB 和 RCA 方法的比较成本分别为 7 美元和 2 美元。
