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Appl Environ Microbiol, July 1998, p. 2639-2643, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Quantification of Toxic Cyanobacteria in Water by Use of Competitive PCR Followed by Sequence-Specific Labeling of Oligonucleotide Probes

Knut Rudi,1,* Olav M. Skulberg,2 Frank Larsen,3 and Kjetill S. Jakobsen1,*

Division of General Genetics, Department of Biology, University of Oslo, Blindern, 0315 Oslo,1 Norwegian Institute for Water Research, Kjelsås, 0411 Oslo,2 and Dynal A/S, Skøyen, 0212 Oslo,3 Norway

Received 22 December 1997/Accepted 24 April 1998

A complete nucleic-acid-based assay which consists of sample preparation, DNA amplification, and chromogenic detection was developed for quantifying potential toxin-producing cyanobacteria of interest to the public. The sample preparation strategy involves the same solid phase for cell concentration and DNA purification. For the detection step, we used a combination of competitive PCR amplification, sequence-specific labeling of oligonucleotide probes, hybridization of the labeled oligonucleotides to immobilized complements and, finally, chromogenic detection. The complete assay was tested with water containing toxin-producing cyanobacteria belonging to the genus Microcystis. A detection limit of 100 cells/ml and a quantitative range of more than 3 orders of magnitude were obtained. This approach can easily be adapted to a wide range of bacterial species and has the potential for simultaneous detection and quantitation of several different target organisms by a single assay.

* Corresponding authors. Mailing address: Division of General Genetics, Department of Biology, University of Oslo, P.O. Box 1031 Blindern, 0315 Oslo, Norway. Phone: 47.22.85.45.73 (Knut Rudi); 47.22.85.46.02 (Kjetill S. Jakobsen). Fax: 47.22.85.46.05. E-mail: knut.rudi{at}bio.uio.no (Knut Rudi); kjetill.jakobsen{at}bio.uio.no (Kjetill S. Jakobsen).

Appl Environ Microbiol, July 1998, p. 2639-2643, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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