2003 Volume 18 Issue 1 Pages 16-23
When module 1 of the microcystin synthetase gene was isolated from two strains, Microcystis aeruginosa NIES298 and M. viridis NIES102 with different types of module 3, the sequence identity was very high. Twenty toxic strains and 18 non-toxic strains of Microcystis were subjected to PCR amplification with primers (M1F3-M1R3) designed from completely conserved regions of module 1. The specific PCR products were amplified from toxic strains and no amplified products were observed in the non-toxic strains. Prior to the development of the competitive PCR, template DNA was isolated from the Microcystis cultures using four isolation methods focused on removing polysaccharide. The potassium xanthogenate-sodium dodecyl sulfate procedure gave a high extraction yield and purity and was used for the preparation of template for competitive PCR. By competitive PCR with M1F3-M1R3, cultured cells of toxic Microcystis were quantitatively detected at 103−105 cells/mL. In environmental water samples from Lake Mikata and Lake Suigetsu, plots of the toxin concentration versus density of toxic cells measured by the competitive PCR showed a positive correlation (r2=0.98), which suggested that competitive PCR with M1F3-M1R3 is useful for the quantitative detection of toxic Microcystis cells in complex environmental samples.