A1 Refereed original research article in a scientific journal
Quantitative PCR detection and improved sample preparation of microcystin-producing Anabaena, Microcystis and Planktothrix
Authors: Hautala H, Lamminmäki U, Spoof L, Nybom S, Meriluoto J, Vehniäinen M
Publisher: ACADEMIC PRESS INC ELSEVIER SCIENCE
Publication year: 2013
Journal: Ecotoxicology and Environmental Safety
Journal name in source: Ecotoxicology and Environmental Safety
Journal acronym: ECOTOX ENVIRON SAFE
Volume: 87
First page : 49
Last page: 56
Number of pages: 8
ISSN: 0147-6513
DOI: https://doi.org/10.1016/j.ecoenv.2012.10.008
Web address : http://www.sciencedirect.com/science/article/pii/S0147651312003600
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.
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.
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