Background: 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. Methods: 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 S. aureus and, then, microbiome was evaluated. Results: 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 S. aureus 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. Conclusions: 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.

Comparison of different conditions for DNA extraction in sputum : A pilot study / M. Oriano, L. Terranova, A. Teri, S. Sottotetti, L. Ruggiero, C. Tafuro, P. Marchisio, A. Gramegna, F. Amati, F. Nava, E. Franceschi, L. Cariani, F. Blasi, S. Aliberti. - In: MULTIDISCIPLINARY RESPIRATORY MEDICINE. - ISSN 2049-6958. - 14:1(2019 Jan 31).

Comparison of different conditions for DNA extraction in sputum : A pilot study

ORIANO, MARTINA;Terranova, Leonardo;Teri, Antonio;SOTTOTETTI, SAMANTHA;Marchisio, Paola;Gramegna, Andrea;Amati, Francesco;NAVA, FABRIZIO;FRANCESCHI, ELISA;Blasi, Francesco;Aliberti, Stefano
2019-01-31

Abstract

Background: 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. Methods: 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 S. aureus and, then, microbiome was evaluated. Results: 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 S. aureus 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. Conclusions: 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.
DNA extraction; Sputum; Microbiota; Microbiome; Sequencing
Settore MED/10 - Malattie dell'Apparato Respiratorio
MULTIDISCIPLINARY RESPIRATORY MEDICINE
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/619431
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