Introduction: There is no consensus on which is the best method to extract bacterial DNA from sputum. We aimed at comparing 20 different DNA extraction techniques on both mock community and sputum samples. Methods: One mock was built and 5 sputa from cystic fibrosis and bronchiectasis patients were evaluated at the Policlinico Hospital in Milan, Italy. 20 techniques for sputum bacterial DNA extraction were compared, including two enzyme-based (Roche and Zymo) and two beads-based (Mobio and QIAGEN) techniques which were tested with/without Dithiothreitol (DTT) and with/without lysostaphin (0.18 and 0.36 mg/mL) steps. Extractions were performed in duplicate to evaluate the variability among samples. DNA was quantified and, then, tested using Real time PCR for 16S rDNA and S. aureus. Results: Although 16S rDNA was similarly detected across all the different techniques in 20 ng of extract (range 19-21 Ct), Roche kit gave the highest DNA yield in both mock and sputa (see figure). The mean standard deviation between replicates was lower in DTT-treated vs non-treated samples (0.17 vs 0.47 Ct, respectively). In both enzymatic-based techniques, the lowest Ct values for Real time PCR for S. aureus in both mock and sputa was identified when lysostaphin was added. Conclusion: An enzyme-based kit with the addition of both DTT treatment and lysostaphin steps seems to be the preferred technique for DNA bacterial extraction on sputum.

What is the best technique to extract bacterial DNA from sputum? / M. Oriano, L. Terranova, L. Ruggiero, C. Tafuro, A. Teri, L. Cariani, E. Franceschi, F. Amati, A. Gramegna, M. Contarini, J. D Chalmers, P.G. Marchisio, S. Aliberti, F.B.A. Blasi. - In: EUROPEAN RESPIRATORY JOURNAL. - ISSN 1399-3003. - 52:suppl. 62(2018 Sep 15), pp. PA5307.1-PA5307.1. ((Intervento presentato al convegno European Respiratory Journal tenutosi a Paris nel 2018.

What is the best technique to extract bacterial DNA from sputum?

M. Oriano;L. Terranova;A. Teri;L. Cariani;E. Franceschi;F. Amati;A. Gramegna;M. Contarini;P.G. Marchisio;S. Aliberti
;
F.B.A. Blasi
2018

Abstract

Introduction: There is no consensus on which is the best method to extract bacterial DNA from sputum. We aimed at comparing 20 different DNA extraction techniques on both mock community and sputum samples. Methods: One mock was built and 5 sputa from cystic fibrosis and bronchiectasis patients were evaluated at the Policlinico Hospital in Milan, Italy. 20 techniques for sputum bacterial DNA extraction were compared, including two enzyme-based (Roche and Zymo) and two beads-based (Mobio and QIAGEN) techniques which were tested with/without Dithiothreitol (DTT) and with/without lysostaphin (0.18 and 0.36 mg/mL) steps. Extractions were performed in duplicate to evaluate the variability among samples. DNA was quantified and, then, tested using Real time PCR for 16S rDNA and S. aureus. Results: Although 16S rDNA was similarly detected across all the different techniques in 20 ng of extract (range 19-21 Ct), Roche kit gave the highest DNA yield in both mock and sputa (see figure). The mean standard deviation between replicates was lower in DTT-treated vs non-treated samples (0.17 vs 0.47 Ct, respectively). In both enzymatic-based techniques, the lowest Ct values for Real time PCR for S. aureus in both mock and sputa was identified when lysostaphin was added. Conclusion: An enzyme-based kit with the addition of both DTT treatment and lysostaphin steps seems to be the preferred technique for DNA bacterial extraction on sputum.
Settore MED/10 - Malattie dell'Apparato Respiratorio
https://erj.ersjournals.com/content/52/suppl_62/PA5307.abstract
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/667573
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