Quantitative Detection of Toxic Strains of the Cyanobacterial Genus Microcystis by Competitive PCR

概要

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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 10^3-10^5 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 (r^2=0.98), which suggested that competitive PCR with M1F3-M1R3 is useful for the quantitative detection of toxic Microcystis cells in complex environmental samples.