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Improved Detection of Bifidobacteria with Optimised 16S rRNA-Gene Based Pyrosequencing

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 o...

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Autores principales: Sim, Kathleen, Cox, Michael J., Wopereis, Harm, Martin, Rocio, Knol, Jan, Li, Ming-Shi, Cookson, William O. C. M., Moffatt, Miriam F., Kroll, J. Simon
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314643/
https://www.ncbi.nlm.nih.gov/pubmed/22470420
http://dx.doi.org/10.1371/journal.pone.0032543
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author Sim, Kathleen
Cox, Michael J.
Wopereis, Harm
Martin, Rocio
Knol, Jan
Li, Ming-Shi
Cookson, William O. C. M.
Moffatt, Miriam F.
Kroll, J. Simon
author_facet Sim, Kathleen
Cox, Michael J.
Wopereis, Harm
Martin, Rocio
Knol, Jan
Li, Ming-Shi
Cookson, William O. C. M.
Moffatt, Miriam F.
Kroll, J. Simon
author_sort Sim, Kathleen
collection PubMed
description 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.
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spelling pubmed-33146432012-04-02 Improved Detection of Bifidobacteria with Optimised 16S rRNA-Gene Based Pyrosequencing Sim, Kathleen Cox, Michael J. Wopereis, Harm Martin, Rocio Knol, Jan Li, Ming-Shi Cookson, William O. C. M. Moffatt, Miriam F. Kroll, J. Simon PLoS One Research Article 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. Public Library of Science 2012-03-28 /pmc/articles/PMC3314643/ /pubmed/22470420 http://dx.doi.org/10.1371/journal.pone.0032543 Text en Sim et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Sim, Kathleen
Cox, Michael J.
Wopereis, Harm
Martin, Rocio
Knol, Jan
Li, Ming-Shi
Cookson, William O. C. M.
Moffatt, Miriam F.
Kroll, J. Simon
Improved Detection of Bifidobacteria with Optimised 16S rRNA-Gene Based Pyrosequencing
title Improved Detection of Bifidobacteria with Optimised 16S rRNA-Gene Based Pyrosequencing
title_full Improved Detection of Bifidobacteria with Optimised 16S rRNA-Gene Based Pyrosequencing
title_fullStr Improved Detection of Bifidobacteria with Optimised 16S rRNA-Gene Based Pyrosequencing
title_full_unstemmed Improved Detection of Bifidobacteria with Optimised 16S rRNA-Gene Based Pyrosequencing
title_short Improved Detection of Bifidobacteria with Optimised 16S rRNA-Gene Based Pyrosequencing
title_sort improved detection of bifidobacteria with optimised 16s rrna-gene based pyrosequencing
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314643/
https://www.ncbi.nlm.nih.gov/pubmed/22470420
http://dx.doi.org/10.1371/journal.pone.0032543
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