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Applied and Environmental Microbiology, April 2004, p. 2373-2382, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2373-2382.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Real-Time Quantitative PCR for Assessment of Abundance of Pseudoalteromonas Species in Marine Samples

Torben L. Skovhus,1 Niels B. Ramsing,1 Carola Holmström,2 Staffan Kjelleberg,2 and Ingela Dahllöf3*

Department of Microbial Ecology, University of Aarhus, Aarhus,1 Department of Marine Ecology, National Environmental Research Institute, Roskilde, Denmark,3 School of Biotechnology and Biomolecular Sciences and Centre of Marine Biofouling and Bio-Innovation, University of New South Wales, Sydney, New South Wales, Australia2

Received 16 September 2003/ Accepted 15 December 2003

A real-time quantitative PCR (RTQ-PCR) method for measuring the abundance of Pseudoalteromonas species in marine samples is presented. PCR primers targeting a Pseudoalteromonas-specific region of the 16S rRNA gene were tested at three different levels using database searches (in silico), a selection of pure cultures (in vitro), and a combined denaturing gradient gel electrophoresis and cloning approach on environmental DNA (in situ). The RTQ-PCR method allowed for the detection of SYBR Green fluorescence from double-stranded DNA over a linear range spanning six orders of magnitude. The detection limit was determined as 1.4 fg of target DNA (1,000 gene copies) measured in the presence of 20 ng of nontarget DNA from salmon testes. In this study, we discuss the importance of robust post-PCR analyses to overcome pitfalls in RTQ-PCR when samples from different complex marine habitats are analyzed and compared on a nonroutine basis. Representatives of the genus Pseudoalteromonas were detected in samples from all investigated habitats, suggesting a widespread distribution of this genus across many marine habitats (e.g., seawater, rocks, macroalgae, and marine animals). Three sample types were analyzed by RTQ-PCR to determine the relative abundance of Pseudoalteromonas ribosomal DNA (rDNA) compared to the total abundance of eubacterial rDNA. The rDNA fractions of Pseudoalteromonas compared to all Eubacteria were 1.55% on the green alga Ulva lactuca, 0.10% on the tunicate Ciona intestinalis, and 0.06% on the green alga Ulvaria fusca.

* Corresponding author. Mailing address: Department of Marine Ecology, National Environmental Research Institute, Frederiksborgvej 399, DK-4000 Roskilde, Denmark. Phone: 45 4630 1317. Fax: 45 4630 1114. E-mail: ind{at}dmu.dk.

Applied and Environmental Microbiology, April 2004, p. 2373-2382, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2373-2382.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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