A Simple and Rapid Method for Detection and Quantification of Microcystin-Producing Cyanobacteria

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  • JIMBO YUSUKE
    1Graduate School of Life and Environmental Science, University of Tsukuba
  • OKANO KUNIHIRO
    1Graduate School of Life and Environmental Science, University of Tsukuba
  • UTSUMI MOTOO
    1Graduate School of Life and Environmental Science, University of Tsukuba
  • SUGIURA NORIO
    1Graduate School of Life and Environmental Science, University of Tsukuba

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  • whole-cell PCRを用いたmicrocystin産生藍藻の簡易・迅速定量法の開発

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Abstract

Toxic Microcystis sp. have been widely recognized as the most common bloom-forming cyanobacteria worldwide. In this study, initial screening method of detection of microcystin-producing Microcystis sp. with simplification, rapidness and sensitiveness was developed, using whole-cell PCR (polymerase chain reaction) and competitive PCR. When whole-cell PCR was carried out on axenic microcystin-producing Microcystis sp., the amplification products were the same size as the products obtained from traditional PCR performed on DNA extracted from Microcystis sp.; and the method was effective in selectively identifying the amplification products from toxic strains. Using the whole-cell method, the target sequence (microcystin synthase gene) could be amplified at least twice as rapidly without DNA extraction, and detection of toxic Microcystis sp. by whole-cell PCR of the target DNA sequence took 1/3 the time required for high-performance liquid chromatography (HPLC) detection of the toxic microcystin. Moreover, the method proved to be sensitive enough to detect toxic cyanobacteria in a sample with less than 10,000 Microcystis cells per ml (about 60 cells per PCR reaction). It was also possible to detect toxic cyanobacteria at in-situ levels as well as in axenic Microcystis strains. Moreover, microcystin-producing Microcystis sp. could be quantified simply by competitive whole-PCR.

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