The identification, selection, and validation of specific qPCR primers that target relevant groups of common gut bacteria and which are able to perform well during a universal thermocycling program was the primary focus of this study. Numerous primer sets targeting different bacterial taxonomical groups including species, genera, and phyla of the gut microbiota have been published during the last decades; however far from all have been evaluated in depth for their specificity to the taxonomical group that they were designed to amplify. Primer validation may be performed either in silico with reference to, for example, the RDP or by laboratory tests against a panel of DNA extracted from related bacteria. In the present study, extracted DNA from a total of 28 microbial species was used for the specificity validation of 58 qPCR primer sets all targeting the 16S rRNA gene of gut bacteria. One universal primer set was included designed to target the V3 variable regions (positions 339–539 in the Escherichia coli gene) of all known bacteria (Walter et al., 2000; Chakravorty et al., 2007). This primer set was shown in silico to match on average 99.1% ± 0.88% of a total of 931 412 good-quality (> 1200 bp) 16S rRNA gene sequences representing Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrucomicrobia, respectively, found in RDP, with allowance for two mismatches. In some cases, unspecific amplification or lack of amplification was observed, which may to some extent be caused by the requirement for primers to perform in the applied universal two-step qPCR, with both annealing and elongation at 60 °C. Following the final screening, a total of 32 primer sets collectively representing the five dominating bacterial phyla of the gut microbiota, as well as the Euryarcheota (Methanobrevibacter smithii) and one universal bacterial primer set, were selected for the GULDA (Table 1). The specificity of these primers was overall consistent with the expected target groups, and amplification efficiencies were comparable to those observed for the universal bacterial primer set, as determined by differences in Ct-values following amplification on pure culture DNA (Fig. 1). It was recently shown that it is possible to optimize qPCR assay efficiency by primer modification, in order to run 16S rRNA gene primers displaying optimal specificities at different annealing temperatures on the same PCR plate under the same experimental conditions (Bacchetti De Gregoris et al., 2011). In the present study, the PCR efficiency for each amplicon group was calculated separately from the slope of the amplification curve by linear regression within the window of linearity (logarithmic scale) by the use of the linregpcr software. The mean calculated efficiencies for each amplicon group were then used to determine the initial concentration, N0, of the DNA target, that is, specific 16S rRNA gene, in arbitrary fluorescence units (Ramakers et al., 2003; Ruijter et al., 2009). All N0-values for specific bacterial taxa were normalized to the calculated universal bacterial N0-value amplified on the same plate. Normalized N0-values obtained from the same amplicon group, for example phylum Bacteroidetes, are directly comparable to each other and may thus be used to determine the fold-change of specific groups of bacteria between two or more samples. Direct comparisons between different amplicon groups may, however, be biased due to, for example, differences in amplicon length and GC-content causing nonequal binding of SYBR Green. Analysis of the fecal samples obtained from six infants at both 9 and 18 months was performed by PCA of normalized N0-values calculated from GULDA (Fig. 2). The score plot shows that all six individuals migrated from left to right along the PC#1 with generally little movement vertically along the PC#2. The initial grouping appeared more confined for the individuals at 9 months as compared to 18 months, which is consistent with the development of a more complex and individual gut microbiota. The loading plot indicates which bacterial taxa drive this migration, which was further studied by calculating the fold-changes for specific amplicon groups (Fig. 3). The latter shows a significant (P < 0.05) increase in the relative abundance of Clostridial cluster IV (Clostridia leptum group) and Bifidobacterium bifidum and concurrent significant decrease in the abundance of Clostridium butyricum (Clostridial cluster I) and a tendency for decrease in Enterobacteriaceae and E. coli (P < 0.10) from 9 to 18 months of age. These findings are consistent with some of the results of a previous study using both qPCR and Northern blotting to characterize intestinal bacteria in infant stools (Hopkins et al., 2005), which showed a significant decrease in Enterobacteria and increase in Faecalibacterium prausnitzii (Clostridial cluster IV) rRNA after 6 months of age. No other significant changes were observed within the remaining 26 amplification groups in the present study. Although a fairly small dataset, n = 6 infants, was used in this study, the methodological protocol of calculating the changes in a microbial community over a time period by the use of relative qPCR determinations performed under universal temperature cycling conditions was successfully demonstrated. In the present study, multiple bifidobacterial species were included in the array as this genus is known to be highly represented during infancy (Roger et al., 2010). Modification of GULDA for other purposes by incorporating other primer sets is, however, easily achieved and provides a high degree of flexibility to the qPCR array. We expect GULDA to be a very useful tool to study induced changes in the composition of the gut microbiota and consequently further elucidate the causal relationships between the vast numbers of bacterial species present in the human gut microbiota.