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首页> 外文期刊>Journal of Clinical Microbiology >Use of a Suspension Array for Rapid Identification of the Varieties and Genotypes of the Cryptococcus neoformans Species Complex
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Use of a Suspension Array for Rapid Identification of the Varieties and Genotypes of the Cryptococcus neoformans Species Complex

机译：悬浮阵列用于快速鉴定新型隐球菌物种复合体的品种和基因型的用途

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Cryptococcus neoformans is an encapsulated fungal pathogen known to cause severe disease in immunocompromised patients. The disease, cryptococcosis, is mostly acquired by inhalation and can result in a chronic meningoencephalitis, which can be fatal. Here, we describe a molecular method to identify the varieties and genotypic groups within the C. neoformans species complex from culture-based assays. The method employs a novel flow cytometer with a dual laser system that allows the simultaneous detection of different target sequences in a multiplex and high-throughput format. The assay uses a liquid suspension hybridization format with specific oligonucleotide probes that are covalently bound to the surface of fluorescent color-coded microspheres. Biotinylated target amplicons, which hybridized to their complementary probe sequences, are quantified by the addition of the conjugate, streptavidin R-phycoerythrin. In this study we developed and validated eight probes derived from sequence analysis of the intergenic spacer region of the rRNA gene region. The assay proved to be specific and sensitive, allowed discrimination of a 1-bp mismatch with no apparent cross-reactivity, and detected 101 to 103 genome copies. The described protocol, which can be used directly with yeast cells or isolated DNA, can be undertaken in less than 1 h following PCR amplification and permits identification of species in a multiplex format. In addition to a multiplex capability, the assay allows the simultaneous detection of target sequences in a single reaction. The accuracy, speed, flexibility, and sensitivity of this technology are a few of the advantages that will make this assay useful for the diagnosis of human cryptococcal infections and other pathogenic diseases.
机译：新型隐球菌是一种封装的真菌病原体，已知会在免疫功能低下的患者中引起严重的疾病。隐球菌性疾病主要通过吸入获得，可导致慢性脑膜脑炎，这可能是致命的。在这里，我们描述了一种分子方法来鉴定 C内的品种和基因型组。新的物种复合物来自基于文化的分析。该方法采用了具有双激光系统的新型流式细胞仪，该系统允许以多重和高通量格式同时检测不同的靶序列。该测定使用液体悬浮液杂交形式，其具有与荧光色码微球表面共价结合的特定寡核苷酸探针。与它们的互补探针序列杂交的生物素化靶标扩增子通过加入缀合物链霉亲和素R-藻红蛋白来定量。在这项研究中，我们开发并验证了八种源自rRNA基因区域的基因间隔区的序列分析的探针。该方法被证明是特异性和灵敏的，可以区分1-bp错配，没有明显的交叉反应，并且可以检测到10 1 至10 3 基因组拷贝。可以直接与酵母细胞或分离的DNA一起使用的上述方案，可以在PCR扩增后不到1小时的时间内进行，并允许以多重形式鉴定物种。除具有多重功能外，该测定还允许在单个反应中同时检测靶序列。该技术的准确性，速度，灵活性和灵敏性是使该测定法可用于诊断人隐球菌感染和其他病原性疾病的一些优势。

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