Microbial diversity in fecal samples depends on DNA extraction method: easyMag DNA extraction compared to QIAamp DNA stool mini kit extraction

Abstract

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®.

Figure 1

Electrophoresis gel of 3 healthy individuals DNA extracts: HC-1, HC-2, HC-3, respectively. Lane 1-3: DNA extracted using easyMag® with 35 μL silica. Lane 4-6: DNA extracted using easyMag® with 140 μL silica. Lane 7–9: DNA extracted using Qiagen method.

Figure 2

Gel electrophoresis of 16S rDNA PCR products run on the 0.2% gel. Lane 1-8: PCR products using DNA extracted by easyMag® with 140 μL silica. Lanes 9-16: PCR products using DNA extracted by Qiagen. Lanes 17-18: positive (Escherichia coli) and negative control, respectively.

Figure 3

DGGE gel pictures show, 16S rDNA PCR products on faecal DNA extracts obtained using easyMag® with140 μL silica and Qiagen methods. Lanes 1, 3, 5, 7, 9, 11, 13, and 15 are 16S rDNA PCR products using DNA extracted by easyMag®. Lanes 2, 4, 6, 8, 10, 12, 14, and 16 are 16S rDNA PCR products using DNA extracted by Qiagen.

Figure 4

DGGE gel pictures show 16S rDNA PCR products of 3 healthy individuals' diluted faecal DNA extracts: 5, 10, 15 and 20 times, respectively. Lanes 1-4, 9-12, 17-20 show 16S rDNA PCR products of diluted DNA extracted using Qiagen. Lanes 5-8, 13-16, 21-24 show16S rDNA PCR products of diluted DNA extracted using easyMag® with 140 μL silica.

Figure 5

DGGE gel pictures show 16S rDNA PCR products of 3 IBD patients' diluted faecal DNA extracts: 5, 10, 15 and 20 times, respectively. Lanes 1-4, 9-12, 17-20 show 16S rDNA PCR products of diluted DNA extracted using Qiagen. Lanes 5-8, 13-16, 21-24 show 16S rDNA PCR products of diluted DNA extracted using easyMag® with 140 μL silica.