title: Influence of DNA isolation on Q-PCR-based quantification of methanogenic Archaea in biogas fermenters.
abstract: Quantitative real-time PCR (Q-PCR) is commonly applied for the detection of certain microorganisms in environmental samples. However, some environments, like biomass-degrading biogas fermenters, are enriched with PCR-interfering substances. To study the impact of the DNA extraction protocol on the results of Q-PCR-based analysis of the methane-producing archaeal community in biogas fermenters, nine different protocols with varying cell disruption and DNA purification approaches were tested. Differences in the quantities of the isolated DNA and the purity parameters were found, with the best cell lysis efficiencies being obtained by a combined lysozyme/SDS-based lysis. When DNA was purified by sephacryl columns, the amount of DNA decreased by one log cycle but PCR inhibitors were eliminated sufficiently. In the case of detection of methanogenic Archaea, the chosen DNA isolation protocol strongly influenced the Q-PCR-based determination of 16S rDNA copy numbers. For example, with protocols including mechanical cell disruption, the 16S rDNA of Methanobacteriales were predominantly amplified (81-90% of the total 16S rDNA copy numbers), followed by the 16S rDNA of Methanomicrobiales (9-18%). In contrast, when a lysozyme/SDS-based cell lysis was applied, the 16S rDNA copy numbers determined for these two orders were the opposite (Methanomicrobiales 82-95%, Methanobacteriales 4-18%). In extreme cases, the DNA isolation method led to discrimination of some groups of methanogens (e.g. members of the Methanosaetaceae). In conclusion, for extraction of high amounts of microbial DNA with high purity from samples of biogas plants, a combined lysozyme/SDS-based cell lysis followed by a purification step with sephacryl columns is recommended.
authors.affiliation: Leibniz-Institut für Agrartechnik Potsdam-Bornim eV, Abteilung Bioverfahrenstechnik, Potsdam, Germany.
authors.forename: I
authors.initials: I
authors.lastname: Bergmann
authors.forename: K
authors.initials: K
authors.lastname: Mundt
authors.forename: M
authors.initials: M
authors.lastname: Sontag
authors.forename: I
authors.initials: I
authors.lastname: Baumstark
authors.forename: E
authors.initials: E
authors.lastname: Nettmann
authors.forename: M
authors.initials: M
authors.lastname: Klocke
mesh_terms: D001105:Archaea / Q000302:isolation & purification*; D019641:DNA, Archaeal / Q000235:genetics / Q000302:isolation & purification*; D004783:Environmental Microbiology; D018628:Gene Dosage; D008697:Methane / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D019641:DNA, Archaeal; D012336:RNA, Ribosomal, 16S; D008697:Methane