A targeted real-time PCR assay for studying naphthalene degradation in the environment.
A quantitative real-time polymerase chain reaction (PCR) assay was developed for monitoring naphthalene degradation during bioremediation processes. The phylogenetic affiliations of known naphthalene-hydroxylating dioxygenase genes were determined to target functionally related bacteria, and degenerate primers were designed on the basis of the close relationships among dioxygenase genes identified from naphthalene-degrading Proteobacteria. Evaluation of the amplification specificity demonstrated that the developed real-time PCR assay represents a rapid, precise means for the group-specific enumeration of naphthalene-degrading bacteria. According to validation with bacterial pure cultures, the assay discriminated between the targeted group of naphthalene dioxygenase sequences and genes in other naphthalene or aromatic hydrocarbon-degrading bacterial strains. Specific amplification of gene fragments sharing a high sequence similarity with the genes included in the assay design was also observed in soil samples recovered from large-scale remediation processes. The target genes could be quantified reproducibly at over five orders of magnitude down to 3 x 10(2) gene copies. To investigate the suitability of the assay in monitoring naphthalene biodegradation, the assay was applied in enumerating the naphthalene dioxygenase genes in a soil slurry microcosm. The results were in good agreement with contaminant mineralization and dot blot quantification of nahAc gene copies. Furthermore, the real-time PCR assay was found to be more sensitive than hybridization-based analysis.
Nyyssönen
Mari
M
VTT Technical Research Center of Finland, P.O. Box 1000, 02044 VTT, Espoo, Finland. mari.nyyssonen@vtt.fi
Piskonen
Reetta
R
Itävaara
Merja
M
D001673:Biodegradation, Environmental; D049308:Dioxygenases; D004784:Environmental Monitoring; D005784:Gene Amplification; D006900:Hydroxylation; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism*; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D020560:Proteobacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012988:Soil Microbiology; D012989:Soil Pollutants / Q000378:metabolism; D013045:Species Specificity
D009097:Multienzyme Complexes; D009281:Naphthalenes; D012989:Soil Pollutants; C031721:naphthalene; D010105:Oxygenases; D049308:Dioxygenases; C038703:naphthalene dioxygenase