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

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.

Impact of DNA extraction method and targeted 16S-rRNA hypervariable region on oral microbiota profiling

“…The supernatant was discarded and the DNA pellets were washed twice with 250 μl of 70% ethanol. Once dried, DNA was dissolved in 100 μl of double-distilled water 46 , 47 .…”

Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex

“…However, in samples positive by both methods, Pneumo4B gave results 3-4 Ct values lower than results of standard PCR ( Supplementary Table 3), suggesting this assay has a higher sensitivity for M. bovis than the reference method. Several factors influence PCR efficiency, such as sample storage and transport, differences in materials (Buzard et al, 2012) and reagents including DNA preparation method (Bowman et al, 2016;Dilhari et al, 2017). These may explain the sensitivity difference between the two methods for M. bovis detection and the moderate agreement of the two assays is also consistent with the Pneumo4B assay being more sensitive than the reference standard reference.…”

Extraction of high molecular weight abaca DNA suitable for next-generation sequencing

“…Protocol 4 as compared to the other methods has the shortest processing time due to absence of a 65 o C incubation (Table 1). Protocols having CTAB as the main detergent for lysis require an incubation time for at least 15 min at 65 o C. This ensures proper lysis of cells and sequestration of proteins and polysaccharides from the DNA [24,25]. Aside from CTAB, SDS is also an effective detergent in lysing plant tissues [18,23,26].…”

“…Aside from CTAB, SDS is also an effective detergent in lysing plant tissues [18,23,26]. For Protocol 5, Triton X-100 was also added aside from CTAB as lysing reagent which is also an effective detergent for lysing cells [25]. into CTAB based extraction buffers is done to absorb the phenols released upon grinding and prevent their oxidation [14,27,28].…”