Quantification of microcystis in a eutrophic lake by simple DNA extraction and SYBR green real-time PCR.

A simple extraction method and real-time PCR with SYBR-Green I were combined to monitor Microcystis 16S rRNA gene (16S rDNA) concentrations over a wide range. A Fast DNA SPIN Kit (MP Biomedicals) was used to extract rDNA quantitatively. Real-time PCR amplified Microcystis rDNA for quantification with the forward primer Micro229f, which was newly designed by us and was highly specific for Microcystis, and the reverse universal primer 342r. The method developed here can detect Microcystis at concentrations as low as 3 cells mL(-1). The rDNA concentration and cell count were highly correlated in the range from 1.2×10(4) to 1.1×10(6) copies mL(-1) and from 9.5×10(2) to 1.0×10(5) cells mL(-1), respectively, in a canal where Microcystis algal blooms occur annually. The Microcystis rDNA concentration in Lake Kasumigaura was measured by the application of our method to water samples collected monthly from April 2004 to March 2006. Microcystis was not detected by microscopy from January to June 2005, except in May, but our method detected 1.0×10(3) to 1.0×10(4) copies mL(-1) of Microcystis rDNA during this period. This result clearly showed that our method is useful for clarifying the annual fluctuation in Microcystis concentration, especially when concentrations are low.