title: Quantitative PCR detection and improved sample preparation of microcystin-producing Anabaena, Microcystis and Planktothrix.
abstract: Blooms of toxic cyanobacteria, associated with illness and mortality in humans and animals, are becoming increasingly common worldwide. The safe use of surface waters for drinking water production and recreation necessitates assessment of toxigenic cyanobacteria. We have developed simple and reliable sample preparation and qPCR methods to detect microcystin-producing strains of three major bloom-forming genera, Anabaena, Microcystis and Planktothrix. The mcyB second thiolation motif, previously not recognized as a potential target for qPCR, was used as a basis for primer and genus-specific probe design. Assay specificity and sensitivity was confirmed with cultured cyanobacterial strains and the effect of different sample preparation methods on quantification was investigated. Sample filtration and cell lysis reduced assay time and resulted in more efficient amplification compared to DNA extraction. Positive correlation (p<0.005) between mcyB copy numbers and microcystin concentrations was observed in environmental samples. The results encourage the use of qPCR in water risk management.
authors.affiliation: Department of Biotechnology, University of Turku, Tykistökatu 6A, 6th floor, FI-20520 Turku, Finland. henna.hautala@utu.fi
authors.forename: Henna
authors.initials: H
authors.lastname: Hautala
authors.forename: Urpo
authors.initials: U
authors.lastname: Lamminmäki
authors.forename: Lisa
authors.initials: L
authors.lastname: Spoof
authors.forename: Sonja
authors.initials: S
authors.lastname: Nybom
authors.forename: Jussi
authors.initials: J
authors.lastname: Meriluoto
authors.forename: Markus
authors.initials: M
authors.lastname: Vehniäinen
mesh_terms: D017033:Anabaena / Q000145:classification / Q000235:genetics; D000818:Animals; D000458:Cyanobacteria / Q000145:classification / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry; D004784:Environmental Monitoring / Q000379:methods*; D052998:Microcystins / Q000378:metabolism; D046931:Microcystis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012308:Risk Management / Q000379:methods
chemical_list: D004269:DNA, Bacterial; D052998:Microcystins; C078588:microcystin