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Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions.
Scientific Reports ( IF 3.8 ) Pub Date : 2019-12-02 , DOI: 10.1038/s41598-019-54608-x
Antonio Rodríguez 1 , Brecht Guillemyn 2 , Paul Coucke 2 , Mario Vaneechoutte 1
Affiliation  

Fungal infections, ranging from superficial to life-threatening infections, represent a major public health problem that affects 25% of the worldwide population. In this context, the study of host-pathogen interactions within the host is crucial to advance antifungal therapy. However, since fungal cells are usually outnumbered by host cells, the fungal transcriptome frequently remains uncovered. We compared three different methods to selectively lyse human cells from in vitro mixes, composed of Candida cells and peripheral blood mononuclear cells. In order to prevent transcriptional modification, the mixes were stored in RNAlater. We evaluated the enrichment of fungal cells through cell counting using microscopy and aimed to further enrich fungal nucleic acids by centrifugation and by reducing contaminant nucleic acids from the host. We verified the enrichment of fungal DNA and RNA through qPCR and RT-qPCR respectively and confirmed that the resulting RNA has high integrity scores, suitable for downstream applications. The enrichment method provided here, i.e., lysis with Buffer RLT followed by centrifugation, may contribute to increase the proportion of nucleic acids from fungi in clinical samples, thus promoting more comprehensive analysis of fungal transcriptional profiles. Although we focused on C. albicans, the enrichment may be applicable to other fungal pathogens.

中文翻译:

核酸富集真菌病原体以研究宿主与病原体的相互作用。

从表面感染到威胁生命的感染,真菌感染是一个重大的公共卫生问题,影响了全球25%的人口。在这种情况下,研究宿主内的宿主-病原体相互作用对于推进抗真菌治疗至关重要。但是,由于真菌细胞通常比宿主细胞多,因此真菌转录组经常未被发现。我们比较了三种不同的方法,从体外混合物中选择性裂解人细胞,该混合物由念珠菌细胞和外周血单核细胞组成。为了防止转录修饰,将混合物储存在RNAlater中。我们通过使用显微镜的细胞计数评估了真菌细胞的富集,旨在通过离心和减少宿主的污染核酸进一步富集真菌核酸。我们分别通过qPCR和RT-qPCR验证了真菌DNA和RNA的富集,并确认所得的RNA具有较高的完整性评分,适用于下游应用。此处提供的富集方法,即用Buffer RLT裂解,然后离心,可能有助于增加临床样品中真菌中核酸的比例,从而促进真菌转录谱的更全面分析。尽管我们专注于白色念珠菌,但富集可能适用于其他真菌病原体。可能有助于增加临床样品中真菌核酸的比例,从而促进真菌转录谱的更全面分析。尽管我们专注于白色念珠菌,但富集可能适用于其他真菌病原体。可能有助于增加临床样品中真菌核酸的比例,从而促进真菌转录谱的更全面分析。尽管我们专注于白色念珠菌,但富集可能适用于其他真菌病原体。
更新日期:2019-12-02
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