TY - JOUR

T1 - Microbial diversity in fecal samples depends on DNA extraction method

T2 - EasyMag DNA extraction compared to QIAamp DNA stool mini kit extraction

AU - Mirsepasi, Hengameh

AU - Persson, Søren

AU - Struve, Carsten

AU - Andersen, Lee O B

AU - Petersen, Andreas M.

AU - Krogfelt, Karen A.

PY - 2014

Y1 - 2014

N2 - Background: There 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). Findings. In 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. Conclusion: QIAamp DNA Stool Mini Kit DNA extracts are optimal for DGGE runs and this extraction method yields a higher amount of DNA compared to easyMag®.

AB - Background: There 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). Findings. In 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. Conclusion: QIAamp DNA Stool Mini Kit DNA extracts are optimal for DGGE runs and this extraction method yields a higher amount of DNA compared to easyMag®.

KW - Denaturing gradient gel electrophoresis

KW - DNA measurement

KW - easyMag®

KW - Fecal DNA extraction

KW - Inflammatory bowel disease

KW - NanoDrop®

KW - QIAamp DNA stool mini kit

KW - Qubit® system

U2 - 10.1186/1756-0500-7-50

DO - 10.1186/1756-0500-7-50

M3 - Journal article

C2 - 24447346

AN - SCOPUS:84892586947

VL - 7

JO - BMC Research Notes

JF - BMC Research Notes

SN - 1756-0500

IS - 1

M1 - 50

ER -