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      The Gut Microbiotassay: a high-throughput qPCR approach combinable with next generation sequencing to study gut microbial diversity

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          Abstract

          Background

          The intestinal microbiota is a complex and diverse ecosystem that plays a significant role in maintaining the health and well-being of the mammalian host. During the last decade focus has increased on the importance of intestinal bacteria. Several molecular methods can be applied to describe the composition of the microbiota. This study used a new approach, the Gut Microbiotassay: an assembly of 24 primer sets targeting the main phyla and taxonomically related subgroups of the intestinal microbiota, to be used with the high-throughput qPCR chip ‘Access Array 48.48′, AA48.48, (Fluidigm®) followed by next generation sequencing. Primers were designed if necessary and all primer sets were screened against DNA extracted from pure cultures of 15 representative bacterial species. Subsequently the setup was tested on DNA extracted from small and large intestinal content from piglets with and without diarrhoea. The PCR amplicons from the 2304 reaction chambers were harvested from the AA48.48, purified, and sequenced using 454-technology.

          Results

          The Gut Microbiotassay was able to detect significant differences in the quantity and composition of the microbiota according to gut sections and diarrhoeic status. 454-sequencing confirmed the specificity of the primer sets. Diarrhoea was associated with a reduced number of members from the genus Streptococcus, and in particular S. alactolyticus.

          Conclusion

          The Gut Microbiotassay provides fast and affordable high-throughput quantification of the bacterial composition in many samples and enables further descriptive taxonomic information if combined with 454-sequencing.

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          Most cited references34

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          Archaea in coastal marine environments.

          E Delong (1992)
          Archaea (archaebacteria) are a phenotypically diverse group of microorganisms that share a common evolutionary history. There are four general phenotypic groups of archaea: the methanogens, the extreme halophiles, the sulfate-reducing archaea, and the extreme thermophiles. In the marine environment, archaeal habitats are generally limited to shallow or deep-sea anaerobic sediments (free-living and endosymbiotic methanogens), hot springs or deep-sea hydrothermal vents (methanogens, sulfate reducers, and extreme thermophiles), and highly saline land-locked seas (halophiles). This report provides evidence for the widespread occurrence of unusual archaea in oxygenated coastal surface waters of North America. Quantitative estimates indicated that up to 2% of the total ribosomal RNA extracted from coastal bacterioplankton assemblages was archaeal. Archaeal small-subunit ribosomal RNA-encoding DNAs (rDNAs) were cloned from mixed bacterioplankton populations collected at geographically distant sampling sites. Phylogenetic and nucleotide signature analyses of these cloned rDNAs revealed the presence of two lineages of archaea, each sharing the diagnostic signatures and structural features previously established for the domain Archaea. Both of these lineages were found in bacterioplankton populations collected off the east and west coasts of North America. The abundance and distribution of these archaea in oxic coastal surface waters suggests that these microorganisms represent undescribed physiological types of archaea, which reside and compete with aerobic, mesophilic eubacteria in marine coastal environments.
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            The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis

            The Ribosomal Database Project (RDP-II) provides the research community with aligned and annotated rRNA gene sequences, along with analysis services and a phylogenetically consistent taxonomic framework for these data. Updated monthly, these services are made available through the RDP-II website (http://rdp.cme.msu.edu/). RDP-II release 9.21 (August 2004) contains 101 632 bacterial small subunit rRNA gene sequences in aligned and annotated format. High-throughput tools for initial taxonomic placement, identification of related sequences, probe and primer testing, data navigation and subalignment download are provided. The RDP-II email address for questions or comments is rdpstaff@msu.edu.
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              Effect of inulin on the human gut microbiota: stimulation of Bifidobacterium adolescentis and Faecalibacterium prausnitzii.

              Prebiotics are food ingredients that improve health by modulating the colonic microbiota. The bifidogenic effect of the prebiotic inulin is well established; however, it remains unclear which species of Bifidobacterium are stimulated in vivo and whether bacterial groups other than lactic acid bacteria are affected by inulin consumption. Changes in the faecal microbiota composition were examined by real-time PCR in twelve human volunteers after ingestion of inulin (10 g/d) for a 16-d period in comparison with a control period without any supplement intake. The prevalence of most bacterial groups examined did not change after inulin intake, although the low G+C % Gram-positive species Faecalibacterium prausnitzii exhibited a significant increase (10.3% for control period v. 14.5% during inulin intake, P=0.019). The composition of the genus Bifidobacterium was studied in four of the volunteers by clone library analysis. Between three and five Bifidobacterium spp. were found in each volunteer. Bifidobacterium adolescentis and Bifidobacterium longum were present in all volunteers, and Bifidobacterium pseudocatenulatum, Bifidobacterium animalis, Bifidobacterium bifidum and Bifidobacterium dentium were also detected. Real-time PCR was employed to quantify the four most prevalent Bifidobacterium spp., B. adolescentis, B. longum, B. pseudocatenulatum and B. bifidum, in ten volunteers carrying detectable levels of bifidobacteria. B. adolescentis showed the strongest response to inulin consumption, increasing from 0.89 to 3.9% of the total microbiota (P=0.001). B. bifidum was increased from 0.22 to 0.63% (P<0.001) for the five volunteers for whom this species was present.
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                Author and article information

                Contributors
                Journal
                BMC Genomics
                BMC Genomics
                BMC Genomics
                BioMed Central
                1471-2164
                2013
                14 November 2013
                : 14
                : 788
                Affiliations
                [1 ]Section for Bacteriology, Pathology and Parasitology, National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, 1870 Frederiksberg C, Denmark
                [2 ]Section for immunology and vaccinology, National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, 1870 Frederiksberg C, Denmark
                [3 ]Department of Informatics and Mathematical Modelling, Technical University of Denmark, Richard Petersens Plads, Building 305, room 126, 2800 Lyngby, Denmark
                [4 ]Danish Genome Institute, Skt. Lucas Kirkeplads 8, 8000 Århus, Denmark
                [5 ]Present address: Chr. Hansen, Bøge Allé 10, 2970 Hørsholm, Denmark
                Article
                1471-2164-14-788
                10.1186/1471-2164-14-788
                3879714
                24225361
                fccd25b1-e1c4-4aab-ad37-c1b9d1a0df33
                Copyright © 2013 Hermann-Bank et al.; licensee BioMed Central Ltd.

                This is an open access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 7 January 2013
                : 14 October 2013
                Categories
                Methodology Article

                Genetics
                access array 48.48,bacteria,intestine,microbiota,qpcr
                Genetics
                access array 48.48, bacteria, intestine, microbiota, qpcr

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