A targeted real-time PCR assay for studying naphthalene degradation in the environment

Mari Nyyssönen (Corresponding Author), Reetta Piskonen, Merja Itävaara

Research output: Contribution to journalArticleScientificpeer-review

22 Citations (Scopus)

Abstract

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 × 102 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.
Original languageEnglish
Pages (from-to)533-543
Number of pages11
JournalMicrobial Ecology
Volume52
Issue number3
DOIs
Publication statusPublished - 2006
MoE publication typeA1 Journal article-refereed

Fingerprint

naphthalene
polymerase chain reaction
quantitative polymerase chain reaction
assay
degradation
gene
assays
genes
amplification
aromatic hydrocarbons
monitoring
Proteobacteria
bacteria
bioremediation
bacterium
remediation
biodegradation
aromatic hydrocarbon
microcosm
mineralization

Keywords

  • bioremediation
  • process monitoring
  • napthalene dioxygenase
  • real-time PCR

Cite this

Nyyssönen, Mari ; Piskonen, Reetta ; Itävaara, Merja. / A targeted real-time PCR assay for studying naphthalene degradation in the environment. In: Microbial Ecology. 2006 ; Vol. 52, No. 3. pp. 533-543.
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A targeted real-time PCR assay for studying naphthalene degradation in the environment. / Nyyssönen, Mari (Corresponding Author); Piskonen, Reetta; Itävaara, Merja.

In: Microbial Ecology, Vol. 52, No. 3, 2006, p. 533-543.

Research output: Contribution to journalArticleScientificpeer-review

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AB - 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 × 102 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.

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