Detection of toxin-producing cyanobacteria by use of paramagnetic beads for cell concentration and DNA purification.
Early detection of water blooms caused by potential toxin-producing cyanobacteria is important in environmental monitoring. We present a new nucleic acid-based method for detection of cyanobacteria in water that utilizes the same paramagnetic solid phase (beads) for both bacterial cell concentration and subsequent DNA purification. In the cell concentration step, the beads were attracted to a magnet after cell adsorption (in an alcohol- and salt-containing solution), and the supernatant was removed. For DNA purification, a buffer containing guanidine thiocyanate and Sarkosyl lysed the concentrated cells. The addition of alcohol precipitated the released DNA onto the same solid phase as was used for the cell concentration. Finally, to remove PCR inhibitors, the DNA was washed twice in alcohol while bound to the beads. All of the bead-DNA complex was used in the subsequent PCR amplification. The detection limit, as measured by 16S rDNA PCR amplification, was 50 cells in a 0.5-ml water sample, which is considerably lower than the limit (500 cells/ml) of toxic cyanobacteria tolerated in drinking water (New South Wales Blue-Green Algae Task Force, 1992). Testing of water from natural habitats showed a detection limit in the same range as that for the defined samples. The detection limits and the simplicity of the method (paramagnetic beads can be handled in automated systems) suggest that our method is suitable for routine environmental monitoring.
Rudi
K
K
Department of Biology, University of Oslo, Norway. knut.rudi@bio.uio.no
Larsen
F
F
Jakobsen
K S
KS
D001331:Automation; D001427:Bacterial Toxins / Q000378:metabolism*; D001431:Bacteriological Techniques; D002458:Cell Fractionation / Q000379:methods*; D000458:Cyanobacteria / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D014871:Water Microbiology; D014881:Water Supply
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S