Abstract
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.
Highlights
Microorganisms associated with polymicrobial infections such as chronic wound infections are diverse and often unculturable (Han et al 2011; Oates et al 2012; Wadinamby 2013)
These extraction methods have been separately reported for deoxyribonucleic acid (DNA) extraction from several biological specimens (Ariefdjohan et al 2010; Oates et al 2012; Walter et al 2001), our study further extends this knowledge by a direct comparison of these methods and application of the extracted DNA to polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) technique
All ten patients included in the study had chronic foot wound infections
Summary
Microorganisms associated with polymicrobial infections such as chronic wound infections are diverse and often unculturable (Han et al 2011; Oates et al 2012; Wadinamby 2013). The goal of this study was to compare the relative efficacy of five conventional DNA extraction methods and one commercial kit in extracting bacterial genomic DNA from infected tissues of chronic wound specimens. These extraction methods have been separately reported for DNA extraction from several biological specimens (Ariefdjohan et al 2010; Oates et al 2012; Walter et al 2001), our study further extends this knowledge by a direct comparison of these methods and application of the extracted DNA to PCR-DGGE technique. Further we aimed to identify the optimized DNA extraction conditions to obtain a high and pure DNA yield from chronic wound specimens in order to effectively profile bacterial communities using PCR-DGGE
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