Effect of DNA extraction and sample preservation method on rumen bacterial population

Maria Luisa Callegari, Paolo Bani, Katerina Fliegerova, Ilma Tapio, Aurelie Bonin, Jakub Mrazek, Alireza Bayat, Johanna Vilkki, Jan Kopečný, Kevin J. Shingfield, Frederic Boyer, Eric Coissac, Pierre Taberlet, R. John Wallace

Risultato della ricerca: Contributo in rivistaArticolo in rivistapeer review

46 Citazioni (Scopus)

Abstract

The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile
Lingua originaleEnglish
pagine (da-a)80-84
Numero di pagine5
RivistaAnaerobe
DOI
Stato di pubblicazionePubblicato - 2014

Keywords

  • Bacterial diversity
  • Bacteroidetes
  • Extracellular DNA
  • Firmicutes
  • Intracellular DNA
  • PCR-DGGE

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