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


RESULTS
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.


CONCLUSIONS
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®.
Department of Microbiology & Infection Control, Statens Serum Institut, Copenhagen DK-2300 S, Denmark. pas@ssi.dk.
Hengameh
H
Mirsepasi
Søren
S
Persson
Carsten
C
Struve
Lee O B
LO
Andersen
Andreas M
AM
Petersen
Karen A
KA
Krogfelt
D000327:Adsorption; D000328:Adult; D001331:Automation; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001433:Bacteriolysis; D044822:Biodiversity; D004269:DNA, Bacterial / Q000302:isolation & purification*; D058645:Denaturing Gradient Gel Electrophoresis; D005243:Feces / Q000382:microbiology*; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000382:microbiology; D007422:Intestines / Q000382:microbiology; D064307:Microbiota / Q000235:genetics*; D008863:Microspheres; D008875:Middle Aged; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D021521:Ribotyping; D012822:Silicon Dioxide; D013056:Spectrophotometry, Ultraviolet / Q000295:instrumentation / Q000379:methods; D055815:Young Adult
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D011933:Reagent Kits, Diagnostic; D012822:Silicon Dioxide