, 2002), the specificity and coverage for rumen Prevotella were confirmed by in silico analysis. Forty sequences of rumen Prevotella 16S rRNA gene including the four characterized species and 26 rumen Bacteroides sequences

were obtained from the GenBank database. The coverage and specificity of two sets of Prevotella genus-level primers, g-Prevo and PreGen4 (Stevenson & Weimer 2007), were tested in silico. The sequences were subjected to multiple alignments using the Selleck Gefitinib program clustalx to identify sequence identities with the primer sets. In addition to the exact match of the primer sequences with the Prevotella and Bacteroides sequences, the presence of consecutive matching sequences at the 3′ ends of the primer was considered to estimate the specificity. Plasmid DNA to be used as the standard in real-time PCR was obtained by cloning of 16S rRNA gene PCR products into Escherichia coli JM109 cells, as described previously (Koike et al., 2007). For click here the species-specific PCR, the 16S rRNA gene fragment of the respective target species (Table 1) was used to prepare the plasmid DNA. The strains used for plasmid preparation were as follows: P. ruminicola

ATCC19189, Ruminococcus flavefaciens ATCC19208, Ruminococcus albus ATCC27210, P. bryantii B14, Fibrobacter succinogenes ATCC19169, Streptococcus bovis ATCC33317, Treponema bryantii ATCC33254, Selenomonas ruminantium ATCC12561, Anaerovibrio lipolytica ATCC33276, Ruminobacter amylophilus ATCC29744, Succinivibrio dextrinosolvens ATCC19716 and Megasphaera elsdenii ATCC25940. The PCR primers used are shown in Table 1. PCR amplification for the quantification of target bacterial 16S rRNA gene was performed using a LightCycler 2.0 system (Roche Applied Science, Penzberg, Germany). The FastStart DNA Master SYBR Green I was used for PCR. The optimal amplification conditions for each all primer pair were achieved with 3.5 mM (final concentration) MgCl2. The reaction mixture in 20 μL of the final

volume contained 2.5 mM MgCl2, 2 μL 10 × Mastermix (containing FastStart Taq DNA polymerase, reaction buffer, dNTP mixture, 1 mM MgCl2 and SYBR Green I dye), 0.5 pmol of each primer and 10 ng template DNA. The thermal profile consisted of denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, annealing at the temperature indicated for the primer pair (Table 1) for 5 s and 72 °C for an appropriate extension time (Table 1). A 10-fold dilution series of the plasmid DNA standard for the respective target bacterial 16S rRNA gene was run along with the samples. Using standard curves obtained from the amplification profile of known concentrations of the plasmid DNA standard, the respective genes were quantified.