Linked Life Data

16094872

Source:http://linkedlifedata.com/resource/pubmed/id/16094872

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
2005-8-12
pubmed:abstractText
Target site inaccessibility represents a significant problem for fluorescent in situ hybridisation (FISH) of 16S rRNA oligonucleotide probes. For this reason, the Clep1156 probe targeting 16S rRNA of the Clostridium leptum phylogenetic subgroup used for dot blot experiments could not be used until now for FISH. Considering that bacteria from the C. leptum subgroup are very abundant in the human faecal microbiota and may play a significant role in host health, we have used unlabelled helper and competitor oligonucleotides to improve the 16S rRNA in situ accessibility and specificity of the Clep1156 probe and applied this approach to enumerate C. leptum bacteria in this ecosystem. Nine C. leptum target strains and five non-target strains were selected to develop and validate the helper-competitor strategy. Depending on the target strains, the use of helpers enhanced the fluorescence intensity signal of Clep1156 from 0.4-fold to 8.4-fold with a mean value of 3.6-fold, switching this probe from the brightness class V-VI (masked sites) to III-IV (accessible sites). The simultaneous use of helper and competitor oligonucleotides with Clep1156 probe allowed the expected specificity without disturbing in situ accessibility. Quantified by FISH combined with flow cytometry, C. leptum bacteria in human faecal samples (n=22) represented 19 +/- 7% of bacteria on average [4.9-37.5]. We conclude that helper oligonucleotides are very useful to circumvent the problem of target site in situ accessibility, especially when probe design is limited to only one 16S rRNA area and that helpers and competitors may be efficiently combined.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0723-2020
pubmed:author
pubmed:issnType
Print
pubmed:volume
28
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
454-64
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Enumeration of bacteria from the Clostridium leptum subgroup in human faecal microbiota using Clep1156 16S rRNA probe in combination with helper and competitor oligonucleotides.
pubmed:affiliation
Institut National de la Recherche Agronomique, Unité d'Ecologie et de Physiologie du Système Digestif, Bâtiment 405, Domaine de Vilvert, 78 352 Jouy-en-Josas, France.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't, Evaluation Studies