Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: Possible consequences for population fingerprinting of complex microbial communities
The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V-3, V-3-V-5, and V-6-V-8 hyper...
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ftunivleuven:oai:lirias.kuleuven.be:123456789/247760 2023-05-15T18:19:31+02:00 Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: Possible consequences for population fingerprinting of complex microbial communities Huys, Geert Vanhoutte, Tom Joossens, Marie Mahious, Amal S De Brandt, Evie Vermeire, Severine Swings, Jean 2008-06 https://lirias.kuleuven.be/handle/123456789/247760 en eng Springer-Verlag Current Microbiology vol:56 issue:6 pages:553-557 https://lirias.kuleuven.be/handle/123456789/247760 0343-8651 gradient gel-electrophoresis dgge analysis copy number rpob gene fermentations microorganisms identification heterogeneity temperature cultivation Description (Metadata) only IT article 2008 ftunivleuven 2017-06-02T19:17:24Z The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V-3, V-3-V-5, and V-6-V-8 hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V-3 and V-3-V-5 primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed. status: published Article in Journal/Newspaper Siberian sturgeon KU Leuven: Lirias |
institution |
Open Polar |
collection |
KU Leuven: Lirias |
op_collection_id |
ftunivleuven |
language |
English |
topic |
gradient gel-electrophoresis dgge analysis copy number rpob gene fermentations microorganisms identification heterogeneity temperature cultivation |
spellingShingle |
gradient gel-electrophoresis dgge analysis copy number rpob gene fermentations microorganisms identification heterogeneity temperature cultivation Huys, Geert Vanhoutte, Tom Joossens, Marie Mahious, Amal S De Brandt, Evie Vermeire, Severine Swings, Jean Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: Possible consequences for population fingerprinting of complex microbial communities |
topic_facet |
gradient gel-electrophoresis dgge analysis copy number rpob gene fermentations microorganisms identification heterogeneity temperature cultivation |
description |
The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V-3, V-3-V-5, and V-6-V-8 hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V-3 and V-3-V-5 primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed. status: published |
format |
Article in Journal/Newspaper |
author |
Huys, Geert Vanhoutte, Tom Joossens, Marie Mahious, Amal S De Brandt, Evie Vermeire, Severine Swings, Jean |
author_facet |
Huys, Geert Vanhoutte, Tom Joossens, Marie Mahious, Amal S De Brandt, Evie Vermeire, Severine Swings, Jean |
author_sort |
Huys, Geert |
title |
Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: Possible consequences for population fingerprinting of complex microbial communities |
title_short |
Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: Possible consequences for population fingerprinting of complex microbial communities |
title_full |
Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: Possible consequences for population fingerprinting of complex microbial communities |
title_fullStr |
Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: Possible consequences for population fingerprinting of complex microbial communities |
title_full_unstemmed |
Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: Possible consequences for population fingerprinting of complex microbial communities |
title_sort |
coamplification of eukaryotic dna with 16s rrna gene-based pcr primers: possible consequences for population fingerprinting of complex microbial communities |
publisher |
Springer-Verlag |
publishDate |
2008 |
url |
https://lirias.kuleuven.be/handle/123456789/247760 |
genre |
Siberian sturgeon |
genre_facet |
Siberian sturgeon |
op_relation |
Current Microbiology vol:56 issue:6 pages:553-557 https://lirias.kuleuven.be/handle/123456789/247760 0343-8651 |
_version_ |
1766196609139343360 |