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Open Access Highly Accessed Methodology article

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

Marie Louise Hermann-Bank1, Kerstin Skovgaard2, Anders Stockmarr3, Niels Larsen4 and Lars Mølbak5*

Author 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

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BMC Genomics 2013, 14:788  doi:10.1186/1471-2164-14-788

Published: 14 November 2013

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.

Keywords:
Access Array 48.48; Bacteria; Intestine; Microbiota; qPCR