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

Abstract

Attached

Infected chronic wounds are polymicrobial in nature which include a diverse group of aerobic and anaerobic microorganisms. Majority of these communal microorganisms are difcult to grow in vitro. DNA fngerprinting methods such as polymerase chain reaction-denaturation gradient gel electrophoresis (PCR-DGGE) facilitate the microbial profling of complex ecosystems including infected chronic wounds. Six diferent DNA extraction methods were compared for profling 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 amplifcation 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 fve DNA extraction methods and gave a broad diversity of bacterial communities in chronic wounds. Among the fve conventional methods, bead beater/ phenol–chloroform based DNA extraction method with STES bufer (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 fndings suggest that the quality and the yield of genomic DNA are infuenced by the DNA extraction protocol, thus a method should be carefully selected in profling a complex microbial community