Comparison of different conditions for DNA extraction in sputum - a pilot study
Comparison of different conditions for DNA extraction in sputum - a pilot study
Authors
Martina Oriano
University of Milan, Department of Pathophysiology and Transplantation, Milano; University of Pavia, Department of Molecular Medicine, Pavia
Leonardo Terranova
Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Internal Medicine Department, Respiratory Unit and Cystic Fibrosis Adult Center, Milano
Antonio Teri
Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Cystic Fibrosis Microbiology Laboratory, Milano
Samantha Sottotetti
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Cystic Fibrosis Microbiology Laboratory, Milano
Luca Ruggiero
Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Paediatric Highly Intensive Care Unit, Milano
Camilla Tafuro
Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Paediatric Highly Intensive Care Unit, Milano
Paola Marchisio
Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Paediatric Highly Intensive Care Unit, Milano
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
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
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
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
Lisa Cariani
Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Cystic Fibrosis Microbiology Laboratory, Milano
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
Stefano Aliberti
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
DNA extraction, Sputum, Microbiota, Microbiome, Sequencing
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.
Oriano M, Terranova L, Teri A, Sottotetti S, Ruggiero L, Tafuro C, et al. Comparison of different conditions for DNA extraction in sputum - a pilot study. Multidiscip Respir Med [Internet]. 2019 Jan. 31 [cited 2024 Jul. 4];14(1). Available from: https://mrmjournal.org/index.php/mrm/article/view/4
We are pleased to announce that we have implemented a new plugin for our journal, which will track citations from Scopus, Google Scholar, Crossref, and Europe PMC. This will allow authors to easily see how many times their work has been cited in these databases. Europe PMC also serves as a search engine and directly links to the work as indexed on PubMed and PubMedCentral, providing additional visibility. The plugin widget (see below) can be found at the bottom of each published work, and by clicking on the database symbols, authors can view the citation numbers.
