Quantification of toxic cyanobacteria in water by use of competitive PCR followed by sequence-specific labeling of oligonucleotide probes.
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.
Rudi
K
K
Department of Biology, University of Oslo, Norway. knut.rudi@bio.uio.no
Skulberg
O M
OM
Larsen
F
F
Jakobsen
K S
KS
D000458:Cyanobacteria / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D014871:Water Microbiology
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes