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Applied and Environmental Microbiology, November 2003, p. 6723-6730, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6723-6730.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Application of Real-Time PCR for Quantification of Microcystin Genotypes in a Population of the Toxic Cyanobacterium Microcystis sp.

Rainer Kurmayer1,2* and Thomas Kutzenberger1

Institute for Limnology, Austrian Academy of Sciences, A-5310 Mondsee, Austria,1 Federal Environmental Agency, D-14195 Berlin, Germany2

Received 23 June 2003/ Accepted 29 August 2003

The cyanobacterium Microcystis sp. frequently develops water blooms consisting of organisms with different genotypes that either produce or lack the hepatotoxin microcystin. In order to monitor the development of microcystin (mcy) genotypes during the seasonal cycle of the total population, mcy genotypes were quantified by means of real-time PCR in Lake Wannsee (Berlin, Germany) from June 1999 to October 2000. Standard curves were established by relating cell concentrations to the threshold cycle (the PCR cycle number at which the fluorescence passes a set threshold level) determined by the Taq nuclease assay (TNA) for two gene regions, the intergenic spacer region within the phycocyanin (PC) operon to quantify the total population and the mcyB gene, which is indicative of microcystin synthesis. In laboratory batch cultures, the cell numbers inferred from the standard curve by TNA correlated significantly with the microscopically determined cell numbers on a logarithmic scale. The TNA analysis of 10 strains revealed identical amplification efficiencies for both genes. In the field, the proportion of mcy genotypes made up the smaller part of the PC genotypes, ranging from 1 to 38%. The number of mcyB genotypes was one-to-one related to the number of PC genotypes, and parallel relationships between cell numbers estimated via the inverted microscope technique and TNA were found for both genes. It is concluded that the mean proportion of microcystin genotypes is stable from winter to summer and that Microcystis cell numbers could be used to infer the mean proportion of mcy genotypes in Lake Wannsee.

* Corresponding author. Mailing address: Austrian Academy of Sciences, Institute for Limnology, Mondseestraße 9, 5310 Mondsee, Austria. Phone: 43 6232 3125 32. Fax: 43 6232 3578. E-mail: rainer.kurmayer{at}oeaw.ac.at.

Applied and Environmental Microbiology, November 2003, p. 6723-6730, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6723-6730.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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