Oriano Martina, Terranova Leonardo, Teri Antonio, Sottotetti Samantha, Ruggiero Luca, Tafuro Camilla, Marchisio Paola, Gramegna Andrea, Amati Francesco, Nava Fabrizio, Franceschi Elisa, Cariani Lisa, Blasi Francesco, Aliberti Stefano
1University of Milan, Department of Pathophysiology and Transplantation, Via Francesco Sforza 35, 20122 Milan, Italy.
3University of Pavia, Department of Molecular Medicine, Pavia, Italy.
Multidiscip Respir Med. 2019 Jan 31;14:6. doi: 10.1186/s40248-018-0166-z. eCollection 2019.
The analysis of microbiome in respiratory samples is a topic of great interest in chronic respiratory diseases. The method used to prepare sputum samples for microbiome analysis is very heterogeneous. The selection of the most suitable methodology for DNA extraction is fundamental to have the most representative data. The objective of this study was to compare different conditions for DNA extraction from sputum in adult patients with bronchiectasis.
Five sputum samples from bronchiectasis patients were collected at the Policlinico Hospital in Milan, Italy. Eighteen conditions for DNA extraction were compared, including two enzyme-based (Roche and Zymo) and one beads-based (Mobio) technique. These techniques were tested with/without Dithiothreitol (DTT) and with/without lysostaphin (0.18 and 0.36 mg/mL) step. DNA was quantified, tested using Real-time PCR for 16S rDNA and and, then, microbiome was evaluated.
Although 16S rDNA was similarly detected across all the different techniques, Roche kit gave the highest DNA yield. The lowest Ct values for Real-time PCR for was identified when lysostaphin was added. Considering genera from microbiome, alpha diversity indices did not show any significant differences between techniques, while relative abundances were more similar in presence of DTT.
None of the conditions emerged to be superior to the others even if enzyme-based kits seem to be needed in order to have a higher extraction yield.
呼吸道样本中的微生物组分析是慢性呼吸道疾病中备受关注的一个话题。用于制备痰液样本以进行微生物组分析的方法非常多样。选择最适合的DNA提取方法对于获得最具代表性的数据至关重要。本研究的目的是比较成年支气管扩张症患者痰液DNA提取的不同条件。
在意大利米兰的Policlinico医院收集了5份支气管扩张症患者的痰液样本。比较了18种DNA提取条件，包括两种基于酶的方法（罗氏和Zymo）和一种基于磁珠的方法（Mobio）。这些方法在有/无二硫苏糖醇（DTT）以及有/无溶葡萄球菌素（0.18和0.36 mg/mL）步骤的情况下进行测试。对DNA进行定量，使用实时PCR检测16S rDNA，然后评估微生物组。
尽管在所有不同方法中均能类似地检测到16S rDNA，但罗氏试剂盒的DNA产量最高。添加溶葡萄球菌素时，实时PCR的最低Ct值被确定。考虑微生物组的属，各方法之间的α多样性指数没有显示出任何显著差异，而在存在DTT的情况下相对丰度更为相似。
即使似乎需要基于酶的试剂盒以获得更高的提取产量，但没有一种条件明显优于其他条件。
