Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal ; Faculty of Sciences, University of Porto, Porto, Portugal.
PLoS One. 2013 Oct 18;8(10):e75968. doi: 10.1371/journal.pone.0075968. eCollection 2013.
Early diagnosis of invasive aspergillosis is essential for positive patient outcome. Likewise genotyping of fungal isolates is desirable for outbreak control in clinical setting. We designed a molecular assay that combines detection, identification, and genotyping of Aspergillus fumigatus in a single reaction.
To this aim we combined 20 markers in a multiplex reaction and the results were seen following mini-sequencing readings. Pure culture extracts were firstly tested. Thereafter, Aspergillus-DNA samples obtained from clinical specimens of patients with possible, probable, or proven aspergillosis according to European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria.
A new set of designed primers allowed multilocus sequence typing (MLST) gene amplification in a single multiplex reaction. The newly proposed SNaPAfu assay had a specificity of 100%, a sensitivity of 89% and detection limit of 1 ITS copy/mL (∼0.5 fg genomic Aspergillus-DNA/mL). The marker A49_F was detected in 89% of clinical samples. The SNaPAfu assay was accurately performed on clinical specimens using only 1% of DNA extract (total volume 50 µL) from 1 mL of used bronchoalveolar lavage.
The first highly sensitive and specific, time- and cost-economic multiplex assay was implemented that allows detection, identification, and genotyping of A. fumigatus strains in a single amplification followed by mini-sequencing reaction. The new test is suitable to clinical routine and will improve patient management.
侵袭性曲霉菌病的早期诊断对患者的预后至关重要。同样，真菌分离株的基因分型对于临床环境中的暴发控制也是理想的。我们设计了一种分子检测方法，可在单次反应中同时检测、鉴定和基因分型烟曲霉。
为此，我们将 20 个标记物组合在一个多重反应中，并通过迷你测序读数来观察结果。首先测试纯培养提取物。然后，根据欧洲癌症研究与治疗组织/真菌病研究组（EORTC/MSG）标准，从可能、可能或确诊为曲霉菌病的患者的临床标本中提取曲霉 DNA 样本。
一组新设计的引物允许在单个多重反应中进行多位点序列分型（MLST）基因扩增。新提出的 SNaPAfu 检测法特异性为 100%，敏感性为 89%，检测限为 1 ITS 拷贝/mL（约 0.5 fg 基因组曲霉-DNA/mL）。在 89%的临床样本中检测到标记物 A49_F。SNaPAfu 检测法在使用 1 毫升支气管肺泡灌洗液中仅提取 50 μL 总 DNA（体积为 50 μL）的情况下，可准确地在临床标本上进行操作。
首次实施了高度敏感和特异性、时间和成本效益高的多重检测方法，可在单次扩增后通过迷你测序反应检测、鉴定和基因分型烟曲霉株。新的检测方法适合临床常规应用，将改善患者的管理。
