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Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method

Infected chronic wounds are polymicrobial in nature which include a diverse group of aerobic and anaerobic microorganisms. Majority of these communal microorganisms are difficult to grow in vitro. DNA fingerprinting methods such as polymerase chain reaction-denaturation gradient gel electrophoresis...

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Autores principales: Dilhari, Ayomi, Sampath, Asanga, Gunasekara, Chinthika, Fernando, Neluka, Weerasekara, Deepaka, Sissons, Chris, McBain, Andrew, Weerasekera, Manjula
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605482/
https://www.ncbi.nlm.nih.gov/pubmed/28929383
http://dx.doi.org/10.1186/s13568-017-0477-z
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author Dilhari, Ayomi
Sampath, Asanga
Gunasekara, Chinthika
Fernando, Neluka
Weerasekara, Deepaka
Sissons, Chris
McBain, Andrew
Weerasekera, Manjula
author_facet Dilhari, Ayomi
Sampath, Asanga
Gunasekara, Chinthika
Fernando, Neluka
Weerasekara, Deepaka
Sissons, Chris
McBain, Andrew
Weerasekera, Manjula
author_sort Dilhari, Ayomi
collection PubMed
description Infected chronic wounds are polymicrobial in nature which include a diverse group of aerobic and anaerobic microorganisms. Majority of these communal microorganisms are difficult to grow in vitro. DNA fingerprinting methods such as polymerase chain reaction-denaturation gradient gel electrophoresis (PCR-DGGE) facilitate the microbial profiling of complex ecosystems including infected chronic wounds. Six different DNA extraction methods were compared for profiling of the microbial community associated with chronic wound infections using PCR-DGGE. Tissue debris obtained from chronic wound ulcers of ten patients were used for DNA extraction. Total nucleic acid was extracted from each specimen using six DNA extraction methods. The yield, purity and quality of DNA was measured and used for PCR amplification targeting V2–V3 region of eubacterial 16S rRNA gene. QIAGEN DNeasy Blood and Tissue Kit (K method) produced good quality genomic DNA compared to the other five DNA extraction methods and gave a broad diversity of bacterial communities in chronic wounds. Among the five conventional methods, bead beater/phenol–chloroform based DNA extraction method with STES buffer (BP1 method) gave a yield of DNA with a high purity and resulted in a higher DGGE band diversity. Although DNA extraction using heat and NaOH had the lowest purity, DGGE revealed a higher bacterial diversity. The findings suggest that the quality and the yield of genomic DNA are influenced by the DNA extraction protocol, thus a method should be carefully selected in profiling a complex microbial community.
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spelling pubmed-56054822017-09-27 Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method Dilhari, Ayomi Sampath, Asanga Gunasekara, Chinthika Fernando, Neluka Weerasekara, Deepaka Sissons, Chris McBain, Andrew Weerasekera, Manjula AMB Express Original Article Infected chronic wounds are polymicrobial in nature which include a diverse group of aerobic and anaerobic microorganisms. Majority of these communal microorganisms are difficult to grow in vitro. DNA fingerprinting methods such as polymerase chain reaction-denaturation gradient gel electrophoresis (PCR-DGGE) facilitate the microbial profiling of complex ecosystems including infected chronic wounds. Six different DNA extraction methods were compared for profiling of the microbial community associated with chronic wound infections using PCR-DGGE. Tissue debris obtained from chronic wound ulcers of ten patients were used for DNA extraction. Total nucleic acid was extracted from each specimen using six DNA extraction methods. The yield, purity and quality of DNA was measured and used for PCR amplification targeting V2–V3 region of eubacterial 16S rRNA gene. QIAGEN DNeasy Blood and Tissue Kit (K method) produced good quality genomic DNA compared to the other five DNA extraction methods and gave a broad diversity of bacterial communities in chronic wounds. Among the five conventional methods, bead beater/phenol–chloroform based DNA extraction method with STES buffer (BP1 method) gave a yield of DNA with a high purity and resulted in a higher DGGE band diversity. Although DNA extraction using heat and NaOH had the lowest purity, DGGE revealed a higher bacterial diversity. The findings suggest that the quality and the yield of genomic DNA are influenced by the DNA extraction protocol, thus a method should be carefully selected in profiling a complex microbial community. Springer Berlin Heidelberg 2017-09-19 /pmc/articles/PMC5605482/ /pubmed/28929383 http://dx.doi.org/10.1186/s13568-017-0477-z Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
spellingShingle Original Article
Dilhari, Ayomi
Sampath, Asanga
Gunasekara, Chinthika
Fernando, Neluka
Weerasekara, Deepaka
Sissons, Chris
McBain, Andrew
Weerasekera, Manjula
Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method
title Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method
title_full Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method
title_fullStr Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method
title_full_unstemmed Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method
title_short Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method
title_sort evaluation of the impact of six different dna extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based dna profiling method
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605482/
https://www.ncbi.nlm.nih.gov/pubmed/28929383
http://dx.doi.org/10.1186/s13568-017-0477-z
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