High-throughput detection of pathogenic yeasts of the genus Trichosporon
High-throughput detection of pathogenic yeasts of the genus Trichosporon
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DOI：
10.1128/jcm.42.8.3696-3706.2004
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发表时间：
2004-08-01
影响因子：
9.4
通讯作者：
Fell, JW
中科院分区：
文献类型：
--
作者：
Diaz, MR;Fell, JW
随着真菌感染发病率的急剧增加，对快速准确检测真菌病原体的方法的需求已变得势在必行。在此，我们测试了Luminex 100，一种新型的流式细胞仪，用于检测医学上重要的丝孢酵母属。该属被选为我们的概念验证模型，由于物种之间的密切系统发育关系。该方法基于核苷酸杂交测定，由共价结合至物种特异性捕获探针的不同荧光珠组的组合组成。杂交后，用635 nm激光通过其光谱地址对携带靶扩增子的珠进行分类。杂交的生物素化扩增子的定量基于用532 nm激光的荧光检测。我们测试了各种多重格式的48个物种特异性和组特异性的捕获探针设计在核糖体DNA的D1/D2区域，内部转录间隔区，和基因间间隔区。用三组引物产生物种特异性生物素化扩增子，以产生来自三个区域的片段。该测定是特异性和快速的，因为它区分了相差1个核苷酸的物种，并且在扩增后处理96孔板需要不到50分钟。该检测试剂盒的灵敏度允许检测PCR中的10（2）个基因组分子和10（7）至10（8）个生物素化扩增产物分子。该技术提供了一种快速检测丝孢酵母属物种的方法，其具有通过组合不同组珠以多重形式鉴定物种的灵活性。
The need for a rapid and accurate method for the detection of fungal pathogens has become imperative as the incidence of fungal infections has increased dramatically. Herein, we tested the Luminex 100, a novel flow cytometer, for the detection of the medically important genus Trichosporon. This genus was selected as our proof-of-concept model due to the close phylogenetic relationship between the species. The method, which is based on a nucleotide hybridization assay, consists of a combination of different sets of fluorescent beads covalently bound to species-specific capture probes. Upon hybridization, the beads bearing the target amplicons are classified by their spectral addresses with a 635-nm laser. Quantitation of the hybridized biotinylated amplicon is based on fluorescence detection with a 532-nm laser. We tested in various multiplex formats 48 species-specific and group-specific capture probes designed in the D1/D2 region of ribosomal DNA, internal transcribed spacer regions, and intergenic spacer region. Species-specific biotinylated amplicons were generated with three sets of primers to yield fragments from the three regions. The assay was specific and fast, as it discriminated species differing by 1 nucleotide and required less than 50 min following amplification to process a 96-well plate. The sensitivity of the assay allowed the detection of 10(2) genome molecules in PCRs and 10(7) to 10(8) molecules of biotinylated amplification product. This technology provided a rapid means of detection of Trichosporon species with the flexibility to identify species in a multiplex format by combining different sets of beads.