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How to Process Sputum Samples and Extract Bacterial DNA for Microbiota Analysis

1
Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Pediatric Highly Intensive Care Unit, 20100 Milan, Italy
2
Department of Clinical Sciences and Community Health, University of Milan, 20100 Milan, Italy
3
Department of Pathophysiology and Transplantation, University of Milan, Internal Medicine Department, Respiratory unit and Adult Cystic Fibrosis Center, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20100 Milan, Italy
4
Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy
5
Cystic Fibrosis Microbiology Laboratory, Fondazione IRCCS Ca’ Granda, 20100 Milan, Italy
6
Department for Sustainability, Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), 00123 Rome, Italy
7
Scottish Centre for Respiratory Research, University of Dundee, Ninewells Hospital and Medical School, DD1 9SY Dundee, UK
*
Author to whom correspondence should be addressed.
These two authors contributed equally and are considered first authors for this manuscript.
Int. J. Mol. Sci. 2018, 19(10), 3256; https://doi.org/10.3390/ijms19103256
Received: 31 August 2018 / Revised: 9 October 2018 / Accepted: 13 October 2018 / Published: 20 October 2018
(This article belongs to the Special Issue Lung Infections: From Molecular Biology to Therapy)
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Abstract

Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for S. aureus. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased S. aureus detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples. View Full-Text
Keywords: DNA extraction; sputum; microbiota; sequencing DNA extraction; sputum; microbiota; sequencing
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Terranova, L.; Oriano, M.; Teri, A.; Ruggiero, L.; Tafuro, C.; Marchisio, P.; Gramegna, A.; Contarini, M.; Franceschi, E.; Sottotetti, S.; Cariani, L.; Bevivino, A.; Chalmers, J.D.; Aliberti, S.; Blasi, F. How to Process Sputum Samples and Extract Bacterial DNA for Microbiota Analysis. Int. J. Mol. Sci. 2018, 19, 3256.

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