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Development and Analysis of qPCR for the Identification of Arthroconidial Yeasts of the Genus Magnusiomyces
Mycopathologia ( IF 3.6 ) Pub Date : 2021-01-03 , DOI: 10.1007/s11046-020-00510-4
Engin Kaplan 1 , Deniz Aktaş 2 , Aylin Döğen 2 , Süleyha Hilmioğlu-Polat 3 , Ramazan Gümral 4 , Ferry Hagen 5, 6, 7 , Macit Ilkit 8 , G Sybren de Hoog 9
Affiliation  

The arthroconidial yeasts Magnusiomyces capitatus and M. clavatus are emerging opportunistic pulmonary pathogens. They are closely related and difficult to distinguish based on morphological and physiological traits. We applied an SYBR® green-based quantitative PCR (qPCR) assay to identify the species. We analyzed 30 reference strains originating from clinical and environmental sources by targeting the Rpb2 gene encoding the second largest subunit of RNA polymerase II. The qPCR assays were tested by direct identification of M. capitatus and M. clavatus in spiked sputum and household dishwasher swabs, respectively, as models for clinical and environmental samples. The assays were proved to be reliable for species-level identification of both species, with 100% sensitivity and 100% specificity, lowest inter-assay deviations (RSDr ≤ 1.65%, R2 values >0.99), detection limit of 10 theoretical copy number of target DNA, and detection cell limit of ≥5000 yeast cells from spiked sputum samples. The developed qPCR assay is a practical molecular approach for the detection of M. capitatus and M. clavatus that can be used as a stand-alone assay or in conjunction with culture-dependent approaches.



中文翻译:

用于鉴定木兰属关节孢子酵母的 qPCR 的开发和分析

节孢子酵母Magnusiomyces capitusM . 棒状杆菌是新兴的机会性肺部病原体。它们关系密切,根据形态和生理特征难以区分。我们应用了基于SYBR ® green 的定量 PCR (qPCR) 分析来鉴定物种。我们通过靶向编码 RNA 聚合酶 II 的第二大亚基的Rpb2基因分析了 30 种来自临床和环境来源的参考菌株。qPCR 测定通过直接鉴定M进行测试。M . 棒状肌分别在加标痰液和家用洗碗机拭子中作为临床和环境样本的模型。该测定被证明对于两种物种的物种水平鉴定是可靠的,具有 100% 的灵敏度和 100% 的特异性,最低的测定间偏差(RSDr ≤ 1.65%,R 2值 >0.99),10 理论拷贝数的检测限目标 DNA 和检测细胞限制≥5000 酵母细胞从加标的痰样本。开发的 qPCR 检测是检测M的实用分子方法。M . clavatus可用作独立检测或与依赖培养的方法结合使用。

更新日期:2021-01-03
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