title: Catalyzed reporter deposition-fluorescent in situ hybridization (CARD-FISH) detection of Dehalococcoides.
abstract: Members of the genus Dehalococcoides are well-known for their capacity to reductively dechlorinate chlorinated organic pollutants. The availability of quantitative and sensitive detection methods is of major interest for research on the ecology of those environmentally important micro-organisms. In this paper we describe the development of a Catalyzed Reporter Deposition-Fluorescent In Situ Hybridization (CARD-FISH) for detection of Dehalococcoides cells in enrichment cultures using two oligonucleotide sequences which target two different lineages of Dehalococcoides as probes. Both sequences were previously applied in conventional FISH as probes. Conjugation of the probe to horseradish peroxidase (HRP) did not change the specificity of the probes and bright fluorescent signals were obtained. Despite the use of higher concentrations of probe and the application of longer exposure times in the conventional FISH procedure, CARD-FISH resulted in more intense signals. The CARD-FISH method was applied to a vinyl chloride (VC)-reductively-dechlorinating enrichment culture. Only the probe targeting the CBDB1 lineage of Dehalococcoides reacted with the sample which was in agreement with previous nucleic acid based analysis. The culture consisted of 51%+/-8% of Dehalococcoides cells. Furthermore, the CARD-FISH probes for detecting Dehalococcoides were combined with FISH probes for simultaneous detection of either Bacteria or Archaea which should allow rapid insight into the relative dynamics of the different members of dechlorinating communities as a response to environmental changes. Overall, CARD-FISH proved to be a rapid, reliable and convenient method to detect and quantify Dehalococcoides cells.
authors.affiliation: Division Soil and Water Management, Catholic University of Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium.
authors.forename: J A
authors.initials: JA
authors.lastname: Dijk
authors.forename: P
authors.initials: P
authors.lastname: Breugelmans
authors.forename: J
authors.initials: J
authors.lastname: Philips
authors.forename: P J
authors.initials: PJ
authors.lastname: Haest
authors.forename: E
authors.initials: E
authors.lastname: Smolders
authors.forename: D
authors.initials: D
authors.lastname: Springael
mesh_terms: D041862:Chloroflexi / Q000235:genetics / Q000302:isolation & purification*; D006735:Horseradish Peroxidase / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
chemical_list: D015345:Oligonucleotide Probes; D006735:Horseradish Peroxidase