Development and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea.

2.50
Hdl Handle:
http://hdl.handle.net/10033/294329
Title:
Development and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea.
Authors:
Labrenz, Matthias; Brettar, Ingrid; Christen, Richard; Flavier, Sebastien; Bötel, Julia; Höfle, Manfred G
Abstract:
We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 x 10(3) to 4.4 x 10(9) copies ml(-1) or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 x 10(1) to 2.2 x10(6) copies ml(-1) or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml(-1). The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.
Affiliation:
Department of Environmental Microbiology, GBF-German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany.
Citation:
Development and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea. 2004, 70 (8):4971-9 Appl. Environ. Microbiol.
Journal:
Applied and environmental microbiology
Issue Date:
Aug-2004
URI:
http://hdl.handle.net/10033/294329
DOI:
10.1128/AEM.70.8.4971-4979.2004
PubMed ID:
15294837
Type:
Article
Language:
en
ISSN:
0099-2240
Appears in Collections:
Publications of RG Environmental Microbiology (UMW)

Full metadata record

DC FieldValue Language
dc.contributor.authorLabrenz, Matthiasen_GB
dc.contributor.authorBrettar, Ingriden_GB
dc.contributor.authorChristen, Richarden_GB
dc.contributor.authorFlavier, Sebastienen_GB
dc.contributor.authorBötel, Juliaen_GB
dc.contributor.authorHöfle, Manfred Gen_GB
dc.date.accessioned2013-06-21T12:39:15Zen
dc.date.available2013-06-21T12:39:15Zen
dc.date.issued2004-08en
dc.identifier.citationDevelopment and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea. 2004, 70 (8):4971-9 Appl. Environ. Microbiol.en_GB
dc.identifier.issn0099-2240en
dc.identifier.pmid15294837en
dc.identifier.doi10.1128/AEM.70.8.4971-4979.2004en
dc.identifier.urihttp://hdl.handle.net/10033/294329en
dc.description.abstractWe have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 x 10(3) to 4.4 x 10(9) copies ml(-1) or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 x 10(1) to 2.2 x10(6) copies ml(-1) or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml(-1). The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.en_GB
dc.language.isoenen
dc.rightsArchived with thanks to Applied and environmental microbiologyen_GB
dc.subject.meshBacteriaen_GB
dc.subject.meshBaltic Statesen_GB
dc.subject.meshColony Count, Microbialen_GB
dc.subject.meshCulture Mediaen_GB
dc.subject.meshDNA, Bacterialen_GB
dc.subject.meshDNA, Ribosomalen_GB
dc.subject.meshPiscirickettsiaceaeen_GB
dc.subject.meshPolymerase Chain Reactionen_GB
dc.subject.meshRNA, Ribosomal, 16Sen_GB
dc.subject.meshSeawateren_GB
dc.subject.meshSensitivity and Specificityen_GB
dc.titleDevelopment and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea.en
dc.typeArticleen
dc.contributor.departmentDepartment of Environmental Microbiology, GBF-German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany.en_GB
dc.identifier.journalApplied and environmental microbiologyen_GB
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