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A Targeted Real-Time PCR Assay for Studying Naphthalene Degradation in the Environment

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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.

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  1. VTT Technical Research Center of Finland, P.O. Box 1000, 02044 VTT, Espoo, Finland

    Mari Nyyssönen, Reetta Piskonen & Merja Itävaara

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  1. Mari Nyyssönen
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  2. Reetta Piskonen
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Correspondence to Mari Nyyssönen.

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Nyyssönen, M., Piskonen, R. & Itävaara, M. A Targeted Real-Time PCR Assay for Studying Naphthalene Degradation in the Environment. Microb Ecol 52, 533–543 (2006). https://doi.org/10.1007/s00248-006-9082-4

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  • DOI: https://doi.org/10.1007/s00248-006-9082-4

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