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

Leonardo Terranova; Martina Oriano; Antonio Teri; Luca Ruggiero; Camilla Tafuro; Paola Marchisio; Andrea Gramegna; Martina Contarini; Elisa Franceschi; Samantha Sottotetti; Lisa Cariani; Annamaria Bevivino; James D. Chalmers; Stefano Aliberti; Francesco Blasi

doi:10.3390/ijms19103256

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

Leonardo Terranova, Martina Oriano, Antonio Teri, Luca Ruggiero, Camilla Tafuro, Paola Marchisio, Andrea Gramegna, Martina Contarini, Elisa Franceschi, Samantha Sottotetti, Lisa Cariani, Annamaria Bevivino, James D. Chalmers, Stefano Aliberti (Lead / Corresponding author), Francesco Blasi

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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.

Original language	English
Article number	3256
Pages (from-to)	1-12
Number of pages	12
Journal	International Journal of Molecular Sciences
Volume	19
Issue number	10
DOIs	https://doi.org/10.3390/ijms19103256
Publication status	Published - 20 Oct 2018

Keywords

DNA extraction
sputum
microbiota
sequencing

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© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Final published version, 4.55 MBLicence: CC BY

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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. (2018). How to Process Sputum Samples and Extract Bacterial DNA for Microbiota Analysis. International Journal of Molecular Sciences, 19(10), 1-12. Article 3256. https://doi.org/10.3390/ijms19103256

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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.",

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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, JD, Aliberti, S & Blasi, F 2018, 'How to Process Sputum Samples and Extract Bacterial DNA for Microbiota Analysis', International Journal of Molecular Sciences, vol. 19, no. 10, 3256, pp. 1-12. https://doi.org/10.3390/ijms19103256

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T1 - How to Process Sputum Samples and Extract Bacterial DNA for Microbiota Analysis

AU - Terranova, Leonardo

AU - Oriano, Martina

AU - Teri, Antonio

AU - Ruggiero, Luca

AU - Tafuro, Camilla

AU - Marchisio, Paola

AU - Gramegna, Andrea

AU - Contarini, Martina

AU - Franceschi, Elisa

AU - Sottotetti, Samantha

AU - Cariani, Lisa

AU - Bevivino, Annamaria

AU - Chalmers, James D.

AU - Aliberti, Stefano

AU - Blasi, Francesco

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N2 - 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.

AB - 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.

KW - DNA extraction

KW - sputum

KW - microbiota

KW - sequencing

U2 - 10.3390/ijms19103256

DO - 10.3390/ijms19103256

M3 - Article

C2 - 30347804

SN - 1661-6596

VL - 19

SP - 1

EP - 12

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

IS - 10

M1 - 3256

ER -

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

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