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
Authors: Chris Sissons
Chinthika Gunasekara
Neluka Fernando
Andrew McBain
Asanga Sampath
Manjula Weerasekera
Deepaka Weerasekara
Ayomi Dilhari
Journal: AMB Express (2017)
Discipline: MICROBIOLOGY
Keywords: DNA extraction methods; Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE); Wound infection.
Affiliations: Department of Microbiology, Faculty of Medical Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka., Department of Surgery, Faculty of Medical Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka., Department of Pathology and Molecular Medicine, University Otago, Wellington, New Zealand., Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, M13 9PT, UK., Department of Microbiology, Faculty of Medical Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka. .

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. 

Retracted: No

PubPeer comments:
 A concern about Figure 3 was brought to my attention by Roger Galligan. * Red boxes: Two lanes showing the results of DNA extraction tissue kit Ka and Kb, respectively, look remarkably similar to each other. * Blue arrows: some bands in the  L lanes and the first two sample lanes (STES+Phen Chl) have a different angle and width than other bands in those lanes. The marked band in left-most L lane appears to have been covered with a sliver of gel or something else. * Not all  L patterns look identical to each other. From the legend it appears that the L samples are a mix of different bacteria or DNAs which would be expected to look the same across all three L lanes. Could the authors please clarify? ![file] , 
 An additional concern about Figure 1. * Green boxes: Three bands - those in lanes K, H1, and S - appear to look very similar to each other. This happens to be the band of interest. * A sharp background transition appears to be visible between lanes BP2 and K. * Image make a bit darker to bring out details. ![file] , 
 Dear Prof Elisabeth M Bik Thank you very much for drawing this to our attention. I am currently investigating by examining the original images and discussing with the researchers involved. Once this is complete we will contact the publisher. Best regards Dr. Manjula Weerasekera , 
 Correction, November 2020, found here: https: amb-express.springeropen.com articles 10.1186 s13568-020-01145-w  Following publication of the original article (Dilhari et al. 2017), the authors identified an error in Figs. 1 and 3. The corrected figures are given below. Could the authors please clarify how two lanes could have been duplicated in error? ,