Application of a real-time PCR assay to a comprehensive method of monitoring harmfula algae

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  • Kamikawa Ryoma
    Laboratory of Marine Microbiology, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University
  • Asai Junko
    Laboratory of Marine Microbiology, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University
  • Miyahara Takahiro
    Shallow Sea Research Institute, Fisheries Experimental Station, Agriculture, Forestry, and Fisheries Research Centre of Oita Prefecture
  • Murata Keisuke
    Division of Fishery Environment, Kagoshima Perfectual Fisheries Technology and Development Centre
  • Oyama Kenichi
    Akashiwo Research Institute of Kagawa Prefecture
  • Yoshimatsu Sadaaki
    Akashiwo Research Institute of Kagawa Prefecture
  • Yoshida Takashi
    Department of Marine Bioscience, Fukui Prefectural University
  • Sako Yoshihiko
    Laboratory of Marine Microbiology, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University

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タイトル別名
  • Application of a Real-time PCR Assay to a Comprehensive Method of Monitoring Harmful Algae

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説明

In this study, we developed a comprehensive method for monitoring representative harmful algal bloom (HAB) species in Japan, namely, three dinoflagellates, Cochlodinium polykrikoides, Karenia mikimotoi, Heterocapsa circularisquama, and four raphidophycean flagellates, Chattonella antiqua, C. marina, C. ovata, and Heterosigma akashiwo; this was done by using a real-time PCR assay with primer sets and probes based on species-specific sequences in the D1/D2 region of 28S rRNA genes. For comprehensive monitoring, a DNA extraction protocol using cetyltrimethylammonium bromide (CTAB) was used. In this quantitative PCR assay, specially designed primer sets and probes showed species-specificity and even 1 cell of each harmful alga was detectable. Detection and quantification of HAB species were unaffected by either the growth phase of the algae or the existence of algae other than the target species in the culture. For environmental samples, this real-time PCR assay could be carried out more rapidly and easily, in addition to the similarity of the cell densities estimated by direct counting under a light microscope and by the real-time PCR assay. Moreover, the real-time PCR assay showed a higher sensitivity in seawater samples in which the harmful algae were not detected by direct counting.<br>

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