Hyperbranched rolling circle amplification as a rapid and sensitive method for species identification within the Cryptococcus species complex
Hyperbranched rolling circle amplification as a rapid and sensitive method for species identification within the Cryptococcus species complex
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DOI：
10.1002/elps.200700903
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发表时间：
2008-08-01
期刊：
影响因子：
2.9
通讯作者：
Meyer, Wieland
中科院分区：
文献类型：
--
作者：
Kaocharoen, Sirada;Wang, Bin;Meyer, Wieland
隐球菌属复合体含有两种密切相关的担子菌酵母：新型隐球菌和格特隐球菌，它们会引起人类和其他动物的隐球菌病。物种和变种具有不同的临床、流行病学、生化和分子特征。目前使用的识别方法要么耗时，要么不再商业化。然而，快速、灵敏和稳健的检测方法来检测这些病原体对于早期诊断和适当的治疗决策至关重要。为了克服这些限制，开发了四种针对 RNA 基因座内转录间隔区 (ITS) 处物种特异性单核苷酸多态性的挂锁探针，并在等温超支化滚环扩增 (HRCA) 过程中应用。这些探针针对 99 个样本进行了测试，其中包括 94 个临床隐球菌培养物、三个密切相关的隐球菌属物种和两个临床标本。挂锁探针的使用以及 HRCA 探针信号放大的组合为准确鉴定新型隐球菌变种提供了快速而灵敏的测定方法。 grubii，C. neoformans var。新型隐球菌和格特隐球菌。当杂合体在 ITS 基因座上杂合时，HRCA 也可用于检测杂合体。 H RCA 结果与之前基于 PCR 指纹、扩增片段长度多态性的基因分型数据一致。和 ITS 测序。
The Cryptococcus species complex contains two closely related basidiomycetous yeasts: Cryptococcus neoformans and C. gattii, which cause cryptococcosis in humans and other animals. The species and varieties are characterized, by different clinical, epidemiological, biochemical and molecular features. The currently used identification methods are either time-consuming or not anymore commercially available. However, a rapid, sensitive and robust assay for the detection of these pathogens is vital for early diagnosis and appropriate treatment decisions. To overcome those limitations, four padlock probes targeting species-specific single nucleotide polymorphisms at the internal transcribed spacers (ITSs) of the RNA gene locus were developed and applied during isothermal hyperbranched rolling circle amplification (HRCA). The probes were tested against 99 samples, including 94 clinical cryptococcal cultures, three closely related Cryptococcus species, and two clinical specimens. The use of the padlock probes and the combination of probe signal amplification by HRCA provided a quick and sensitive assay for the accurate identification of C. neoformans var. grubii, C. neoformans var. neoformans and C. gattii. HRCA was also useful to detect hybrids, when they were heterozygous at the ITS locus. The H RCA results were in agreement with previous genotyping data based on PCR fingerprinting, amplified fragment length polymorphism. and ITS sequencing.