Relative quantification real-time PCR was used to quantify several bacterial species in ruminal samples from two lactating cows, each sampled 3 h after feeding on two successive days. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and eubacterial domain-level primers. Bacterial populations showed a clear predominance of members of the genus Prevotella, which comprised 42% to 60% of the bacterial rRNA gene copies in the samples. However, only 2% to 4% of the bacterial rRNA gene copies were represented by the classical ruminal Prevotella species Prevotella bryantii, Prevotella ruminicola and Prevotella brevis. The proportion of rRNA gene copies attributable to Fibrobacter succinogenes, Ruminococcus flavefaciens, Selenomonas ruminantium and Succinivibrio dextrinosolvens were each generally in the 0.5% to 1% range. Proportions for Ruminobacter amylophilus and Eubacterium ruminantium were lower (0.1% to 0.2%), while Butyrivibrio fibrisolvens, Streptococcus bovis, Ruminococcus albus and Megasphaera elsdenii were even less abundant, each comprising <0.03% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus.
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This work was supported by the Agricultural Research Service through the CRIS project 3655-21000-033-00D. We thank R. Zeltwanger for supplying pure cultures of the bacterial strains, D.R. Mertens and M.B. Hall for the valuable discussions.
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An erratum to this article can be found online at http://dx.doi.org/https://doi.org/10.1007/s00253-009-2033-5.
Sample calculation of the relative population size using real-time PCR
The ratio of the amounts of one target sequence to another, first described by Pfaffl (2001), is given by equation A1.
where E is the efficiency of the PCR reaction and ΔCt is the difference in the number of cycles to crossing threshold. In the example here, the target is the sequence amplified by EubRum2, designed to be species-specific for E. ruminantium. The reference is the sequence amplified by BACset1, specific to the entire domain bacteria. Calculations are provided here for a single rumen sample designated R1.
Two PCR plates were run, one using the primer set EubRum1 and the other using BACset1. The actual data for these two plates are shown in Table A1 (EubRum1) and Table A2 (BACset1). Note that in both plates, the same DNA sample was used at the same series of concentrations. The varying concentrations for the E. ruminantium GA195 DNA was produced by fivefold serial dilutions. The amount of DNA loaded was estimated spectrophotometrically, but relative quantification assures that any inaccuracies in this estimate of the DNA amount do not affect the accuracy of the final ratio results. Only the mass relationships between the serial dilutions are critical and then only for the construction of the line used for calculation of PCR efficiency.
The efficiency values for the EubRum2 and BACT1 primers sets, calculated as the antilog of the negative reciprocal of the line slope, were 1.968 and 1.932, respectively, near the theoretical maximum of 2.0 (i.e., a doubling of the target DNA in each PCR cycle).
An essential assumption is made at this point: that the number of species-specific targets per cell is equal to the number of domain-level primer sites. This allows data from both plates to be compared to one another. An additional assumption is that the efficiency of the primers is the same for DNA isolated from pure cultures and for DNA isolated from rumen samples. Separate experiments have confirmed this.
As per equation A1, relative quantification requires both the amplification efficiency and a control Ct for each sample. Selection of the control Ct can be made at any nominal DNA concentration, as long as the same concentration is selected for both plates. In general, there is less variation among analytical replicates at higher DNA concentrations. Calculations using the most concentrated standard (viz., 20 ng) provides mean Ct values for E. ruminantium of 10.09 (Table A1) and 11.40 (Table A2). The mean Ct values for the unknown rumen sample R1 were 19.42 (Table A1) and 11.31 (Table A2). Applying these values to equation A1, the mean Ct was subtracted from the control Ct:
The efficiency of each reaction, from Fig. A1, was then raised to the power of the calculated value, (control Ct)–(unknown sample Ct). Thus, the numerator of the ratio (for the E. ruminantium-specific primer, plate 1) was (1.968)−9.33 = 0.001806 and the denominator of the ratio, (for the domain bacteria primer, plate 2) was (1.932)0.09 = 1.0611. The ratio in equation A1 can thus be calculated as (0.001806) / (1.0611) = 0.00170, or 0.170%. This represents the ratio of the total E. ruminantium 16S rRNA gene copies to the total bacterial 16S rRNA gene copies for the unknown rumen sample R1.
To verify that the relative quantification method is robust across a wide range of target concentrations, repeating the above calculation with the most dilute of the standards (nominal 0.00128 ng DNA) yields a ratio of 0.00162 (vs 0.00170 for the most concentrated standard).
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Stevenson, D.M., Weimer, P.J. Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR. Appl Microbiol Biotechnol 75, 165–174 (2007). https://doi.org/10.1007/s00253-006-0802-y
- Real-time PCR