Evaluation of different DNA sampling techniques for the application of the real-time PCR method for the quantification of cyanobacteria in water
Article first published online: 10 FEB 2006
Letters in Applied Microbiology
Volume 42, Issue 4, pages 412–417, April 2006
How to Cite
Schober, E. and Kurmayer, R. (2006), Evaluation of different DNA sampling techniques for the application of the real-time PCR method for the quantification of cyanobacteria in water. Letters in Applied Microbiology, 42: 412–417. doi: 10.1111/j.1472-765X.2006.01857.x
- Issue published online: 8 MAR 2006
- Article first published online: 10 FEB 2006
- 2005/0712: received 22 June 2005, revised 19 September 2005 and accepted 20 September 2005
- early warning;
- genotype composition;
- quantitative PCR;
- Taq nuclease assay;
- water monitoring
Aims: To evaluate different types of sample storage and DNA extraction techniques for the real-time PCR quantification of cyanobacteria in water.
Methods and Results: Two different filter types for the cell harvest of Microcystis sp. and Planktothrix spp. that were either freeze-dried or stored frozen, and two different methods for DNA extraction were compared. DNA extraction was achieved by standard phenol-chloroform extraction or by a faster commercially available purification kit (DNeasy®, QIAGEN). In general there was good agreement between the cell number equivalents of phycocyanin (PC) genotypes that were estimated using the Taq nuclease assay (TNA) between both filter types and the storing of samples. The standard DNA extraction procedure gave higher numbers of PC genotypes when compared with the DNeasy procedure. TNA results obtained from Planktothrix from natural samples extracted with the standard procedure revealed a significant correlation with the cell numbers estimated via the microscope.
Conclusions: Freeze-drying of samples gives quantifiable data. The standard DNA extraction is considered to be the most reliable and accurate, although the DNeasy procedure is useful for early warning monitoring.
Significance and Impact of the Study: Application of quantitative genotype analysis in cyanobacteria from freeze-dried samples collected during recent and past sampling programmes.