title: Improved detection of bifidobacteria with optimised 16S rRNA-gene based pyrosequencing.
abstract: The 16S rRNA gene is conserved across all bacteria and as such is routinely targeted in PCR surveys of bacterial diversity. PCR primer design aims to amplify as many different 16S rRNA gene sequences from as wide a range of organisms as possible, though there are no suitable 100% conserved regions of the gene, leading to bias. In the gastrointestinal tract, bifidobacteria are a key genus, but are often under-represented in 16S rRNA surveys of diversity. We have designed modified, 'bifidobacteria-optimised' universal primers, which we have demonstrated detection of bifidobacterial sequence present in DNA mixtures at 2% abundance, the lowest proportion tested. Optimisation did not compromise the detection of other organisms in infant faecal samples. Separate validation using fluorescence in situ hybridisation (FISH) shows that the proportions of bifidobacteria detected in faecal samples were in agreement with those obtained using 16S rRNA based pyrosequencing. For future studies looking at faecal microbiota, careful selection of primers will be key in order to ensure effective detection of bifidobacteria.
authors.affiliation: Imperial College London, St. Mary's Campus, London, United Kingdom. k.sim@imperial.ac.uk
authors.forename: Kathleen
authors.initials: K
authors.lastname: Sim
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Cox
authors.forename: Harm
authors.initials: H
authors.lastname: Wopereis
authors.forename: Rocio
authors.initials: R
authors.lastname: Martin
authors.forename: Jan
authors.initials: J
authors.lastname: Knol
authors.forename: Ming-Shi
authors.initials: MS
authors.lastname: Li
authors.forename: William O C M
authors.initials: WO
authors.lastname: Cookson
authors.forename: Miriam F
authors.initials: MF
authors.lastname: Moffatt
authors.forename: J Simon
authors.initials: JS
authors.lastname: Kroll
mesh_terms: D001644:Bifidobacterium / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D005243:Feces / Q000382:microbiology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007223:Infant; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S