Development and evaluation of quantitative real-time PCR analysis for monitoring the biodegradation of naphthalene

Recent advances in molecular biology have established widespread interest in the development of non-cultivation based techniques suitable for environmental analysis. In bioremediation considerable attention has focused especially on the detection of degradative genes that have been shown to be good indicators of contaminant degradation. However, methods are needed for the assessment of diverse degradative genotypes in the environment. Here we report the application of quantitative real-time PCR analysis for the detection of different naphthalene-degrading bacteria in environmental samples and monitoring the biodegradation process. Several nahA-like genes coding for the large subunit of naphthalene dioxygenase were aligned in order to design degenerated primers, and detection was evaluated in a biodegradation process simulated in Pseudomonas putida G7 inoculated soil slurry and non-inoculated soil slurry. In order to identify the amplified genes, PCR products were separated on DGGE gel and sequenced. A specific PCR product was successfully amplified both in P. putida G7 inoculated soil slurry and in non-inoculated soil slurry. Changes in the number of gene copies were detected during the course of the study, and the quantification results were found to correlate with other monitoring data. Sequence diversity between amplicons was confirmed by PCR-DGGE analysis, and the retrieved sequences were shown to match those used for primer design. According to our results, quantitative real-time PCR is a potential tool for environmental analysis. With careful primer design, gene assays for the specific detection of functional genes can be developed. Financial support was received from Ekokem Foundation and VTT Clean World research program.