title: The currently used commercial DNA-extraction methods give different results of clostridial and actinobacterial populations derived from human fecal samples.
abstract: Recently several human health-related microbiota studies have had partly contradictory results. As some differences may be explained by methodologies applied, we evaluated how different storage conditions and commonly used DNA-extraction kits affect bacterial composition, diversity, and numbers of human fecal microbiota. According to our results, the DNA-extraction did not affect the diversity, composition, or quantity of Bacteroides spp., whereas after a week's storage at -20 °C, the numbers of Bacteroides spp. were 1.6-2.5 log units lower (P < 0.05). Furthermore, the numbers of predominant bacteria, Eubacterium rectale (Erec)-group, Clostridium leptum group, bifidobacteria, and Atopobium group were 0.5-4 log units higher (P < 0.05) after mechanical DNA-extraction as detected with qPCR, regardless of storage. Furthermore, the bacterial composition of Erec-group differed significantly after different DNA-extractions; after enzymatic DNA-extraction, the most prevalent genera detected were Roseburia (39% of clones) and Coprococcus (10%), whereas after mechanical DNA-extraction, the most prevalent genera were Blautia (30%), Coprococcus (13%), and Dorea (10%). According to our results, rigorous mechanical lysis enables detection of higher bacterial numbers and diversity from human fecal samples. As it was shown that the results of clostridial and actinobacterial populations are highly dependent on the DNA-extraction methods applied, the use of different DNA-extraction protocols may explain the contradictory results previously obtained.
authors.affiliation: VTT, Biotechnology, Espoo, Finland. johanna.maukonen@vtt.fi
authors.forename: Johanna
authors.initials: J
authors.lastname: Maukonen
authors.forename: Catarina
authors.initials: C
authors.lastname: Simões
authors.forename: Maria
authors.initials: M
authors.lastname: Saarela
mesh_terms: D000328:Adult; D015373:Bacterial Typing Techniques / Q000379:methods*; D001439:Bacteroides / Q000145:classification / Q000235:genetics; D001483:Base Sequence; D001644:Bifidobacterium / Q000235:genetics; D003013:Clostridium / Q000145:classification* / Q000235:genetics; D004247:DNA; D004269:DNA, Bacterial / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D005051:Eubacterium / Q000145:classification / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D004269:DNA, Bacterial; D004247:DNA