Comparison of different conditions for DNA extraction in sputum - a pilot study

  • Martina Oriano University of Milan, Department of Pathophysiology and Transplantation, Milano; University of Pavia, Department of Molecular Medicine, Pavia, Italy.
  • Leonardo Terranova Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Internal Medicine Department, Respiratory Unit and Cystic Fibrosis Adult Center, Milano, Italy.
  • Antonio Teri Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Cystic Fibrosis Microbiology Laboratory, Milano, Italy.
  • Samantha Sottotetti Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Cystic Fibrosis Microbiology Laboratory, Milano, Italy.
  • Luca Ruggiero Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Paediatric Highly Intensive Care Unit, Milano, Italy.
  • Camilla Tafuro Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Paediatric Highly Intensive Care Unit, Milano, Italy.
  • Paola Marchisio Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Paediatric Highly Intensive Care Unit, Milano, Italy.
  • Andrea Gramegna University of Milan, Department of Pathophysiology and Transplantation, Milano; Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Internal Medicine Department, Respiratory Unit and Cystic Fibrosis Adult Center, Milano, Italy.
  • Francesco Amati University of Milan, Department of Pathophysiology and Transplantation, Milano; Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Internal Medicine Department, Respiratory Unit and Cystic Fibrosis Adult Center, Milano, Italy.
  • Fabrizio Nava University of Milan, Department of Pathophysiology and Transplantation, Milano; Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Internal Medicine Department, Respiratory Unit and Cystic Fibrosis Adult Center, Milano, Italy.
  • Elisa Franceschi University of Milan, Department of Pathophysiology and Transplantation, Milano; Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Internal Medicine Department, Respiratory Unit and Cystic Fibrosis Adult Center, Milano, Italy.
  • Lisa Cariani Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Cystic Fibrosis Microbiology Laboratory, Milano, Italy.
  • Francesco Blasi University of Milan, Department of Pathophysiology and Transplantation, Milano; Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Internal Medicine Department, Respiratory Unit and Cystic Fibrosis Adult Center, Milano, Italy.
  • Stefano Aliberti | stefano.aliberti@unimi.it University of Milan, Department of Pathophysiology and Transplantation, Milano; Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Internal Medicine Department, Respiratory Unit and Cystic Fibrosis Adult Center, Milano, Italy.

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.

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Published
2019-01-31
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Original Research Articles
Keywords:
DNA extraction, Sputum, Microbiota, Microbiome, Sequencing
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How to Cite
Oriano, M., Terranova, L., Teri, A., Sottotetti, S., Ruggiero, L., Tafuro, C., Marchisio, P., Gramegna, A., Amati, F., Nava, F., Franceschi, E., Cariani, L., Blasi, F., & Aliberti, S. (2019). Comparison of different conditions for DNA extraction in sputum - a pilot study. Multidisciplinary Respiratory Medicine, 14(1). https://doi.org/10.4081/mrm.2019.4