Abstract
BackgroundThere are challenges, when extracting bacterial DNA from specimens for molecular diagnostics, since fecal samples also contain DNA from human cells and many different substances derived from food, cell residues and medication that can inhibit downstream PCR. The purpose of the study was to evaluate two different DNA extraction methods in order to choose the most efficient method for studying intestinal bacterial diversity using Denaturing Gradient Gel Electrophoresis (DGGE).FindingsIn this study, a semi-automatic DNA extraction system (easyMag®, BioMérieux, Marcy I’Etoile, France) and a manual one (QIAamp DNA Stool Mini Kit, Qiagen, Hilden, Germany) were tested on stool samples collected from 3 patients with Inflammatory Bowel disease (IBD) and 5 healthy individuals.DNA extracts obtained by the QIAamp DNA Stool Mini Kit yield a higher amount of DNA compared to DNA extracts obtained by easyMag® from the same fecal samples. Furthermore, DNA extracts obtained using easyMag® seemed to contain inhibitory compounds, since in order to perform a successful PCR-analysis, the sample should be diluted at least 10 times. DGGE performed on PCR from DNA extracted by QIAamp DNA Stool Mini Kit DNA was very successful.ConclusionQIAamp DNA Stool Mini Kit DNA extracts are optimal for DGGE runs and this extraction method yields a higher amount of DNA compared to easyMag®.
Highlights
There are challenges, when extracting bacterial DNA from specimens for molecular diagnostics, since fecal samples contain DNA from human cells and many different substances derived from food, cell residues and medication that can inhibit downstream Polymerase chain reaction (PCR)
Results of the DNA measurements (Qubit®) and the density of bands in gel electrophoresis showed a relatively low amount of DNA extracted by easyMag® compared to DNA extracted by QIAamp DNA stool mini kit (Qiagen)
Our results demonstrate that easyMag® can extract DNA from fecal samples and the method is suitable for extracting DNA from fecal samples of Inflammatory Bowel Disease (IBD) patients
Summary
There are challenges, when extracting bacterial DNA from specimens for molecular diagnostics, since fecal samples contain DNA from human cells and many different substances derived from food, cell residues and medication that can inhibit downstream PCR. The purpose of the study was to evaluate two different DNA extraction methods in order to choose the most efficient method for studying intestinal bacterial diversity using Denaturing Gradient Gel Electrophoresis (DGGE). When using Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rDNA it has been demonstrated that the diversity of the microbiota in patients with Inflammatory Bowel Disease (IBD) is less complex than in healthy subjects [14]; the influence of DNA extraction methods is unknown. PCR-DGGE was applied on the DNA extracts from both extraction procedures in order to evaluate the efficiency of the two extraction methods for determining the bacterial diversity in fecal samples from IBD patients and from healthy controls
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