Development of a Quantification Method of Harmful Raphidophytes Chattonella marina and Heterosigma akashiwo by the quantitative PCR assay

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  • Mukai Koki
    Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University
  • Ohta Kohei
    Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University | Laboratory of Marine Biology, Faculty of Agriculture, Kyushu University : Associate Professor
  • Shimasaki Yohei
    Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University : Associate Professor
  • Unoki-Kato Yoko
    Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University
  • Oshima Yuji
    Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University : Professor

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Other Title
  • 定量PCR を用いた有害ラフィド藻Chattonella marina およびHeterosigma akashiwo 定量法の検討
  • テイリョウ PCR オ モチイタ ユウガイ ラフィド ソウ Chattonella marina オヨビ Heterosigma akashiwo テイリョウホウ ノ ケントウ

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We developed a quantification method of harmful raphidophytes Chattonella marina and Heterosigma akashiwo using quantitative PCR from seawater sample. Primers and probes were designed from cyt c region of mtDNA in C. marina and ITS region of rRNA in H. akashiwo as target genes for quantitative PCR. High PCR amplification efficiency (80.0% in C. marina and 82.6% in H. akashiwo) and R^2 value (0.991 in C. marina and 0.998 in H. akashiwo) between Ct value and gene copy number were observed in quantitative PCR using dilution series of the target gene fragments from 103 to 109 copies. Then, we confirmed the species selectivity of the designed primers and probes by quantitative PCR using DNA samples extracted from other algal species. In addition, we checked the accuracy of quantitative value of present method including extraction using DNeasy Plant Mini Kit (QIAGEN) using cultured algal cells. As a result, non-specific gene amplification was not detected in DNA samples extracted from other algal species tested. Also, there were significant relation between quantitative values of cell densities by microscopic observation and quantitative PCR in C. marina (R^2 = 0 . 84 ; p< 0 . 05 ) and H. akashiwo (R^2 =0.96; p<0.05) through the growth phase. These results strongly suggested the application of the present method to field monitoring of C. marina and H. akashiwo.

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