Total bacterial and species-specific 16S rDNA micro-array quantification of complex samples

Sammendrag

Aims: We describe a novel DNA-micro-array-based method that targets 16SrDNA to quantify changes in both the total bacterial DNA and the species-specific DNA composition. Methods and Results: Quantifications were achieved by combining competitive PCR for quantitying total bacterial DNA with quantification of specics-specific DNA composition based on signature 16S rDNA sequences. We constructed 11 different probes, which were evaluated on 21 different strains, in addition to complex samples. The signals obtained with sequence-specific labelling of the probes corresponded well with what should be expected based on 16S rDNA phylogenetic reconstruction. The quantification of species-specific DNA composition showed that the micro-array approach could be used to accurately determine differential growth of bacteria in mixed samples. We analysed samples containing mixtures of Lactococcus lactis and different species of propionibacteria during a 2-week incubation period. Lactococcus lactis grew fast, reaching a maximum after 12 h, Propionibacterium acidipropionici and Propionibacterium freudenreichii reached a maximum after 48 h, whereas Propionibacterium jensenii showed a slow increase during the whole growth period. The 16S rDNA total bacterial DNA quantification was compared with real-time PCR, absorbance measurements (ABS(600)) and colony forming units (CFU). Conclusion: The accuracy of the array approach was in the same range or better than the alternative techniques. The potential of the 16S rDNA micro-array method was further demonstrated using a liquid cheese model. Significance and Impact of the Study: This is to our knowledge the first time quantification of the total bacterial DNA and the species-specific DNA compositions of mixed populations have been achieved in the same assay.