Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis.
The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces is laborious and is one of the bottlenecks holding up the application of microarray and other DNA-based techniques in large cohort studies. In order to enhance the DNA extraction step we combined mechanical disruption of microbial cells by repeated bead-beating (RBB) with two automated DNA extraction methods, KingFisher with InviMag Stool DNA kit (KF) and NucliSENS easyMAG (NeM). The semi-automated DNA extraction methods, RBB combined with either KF or NeM, were compared to the manual extraction method currently considered the most suited method for fecal DNA extraction by assessing the yield of 16S rRNA gene copies by qPCR and total microbiota composition by the HITChip, a phylogenetic microarray. Parallel DNA extractions from infant fecal samples by using the three methods showed that the KF and manual methods gave comparable yields of 16S rRNA gene copies as assessed by qPCR, whereas NeM showed a significantly lower yield. All three methods showed highly similar microbiota profiles in HITChip. Both KF and NeM were found to be suitable methods for DNA extraction from fecal samples after the mechanical disruption of microbial cells by bead-beating. The semi-automated methods could be performed in half of the time required for the manual protocol, while being comparable to the manual method in terms of reagent costs.
Nylund
Lotta
L
Functional Foods Forum, University of Turku, Finland.
Heilig
Hans G H J
HG
Salminen
Seppo
S
de Vos
Willem M
WM
Satokari
Reetta
R
D001331:Automation / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology*; D006801:Humans; D007223:Infant; D054892:Metagenome / Q000235:genetics*; D056186:Metagenomics / Q000379:methods*; D046228:Microarray Analysis / Q000379:methods; D008828:Microbiological Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013048:Specimen Handling / Q000379:methods
D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D004247:DNA