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

向井, 幸樹  ,  太田, 耕平  ,  島崎, 洋平  ,  鵜木(加藤), 陽子  ,  大嶋, 雄治

72 ( 2 )  , pp.39 - 46 , 2017-09-08 , 九州大学大学院農学研究院
定量PCR を用いて,海水中に存在する有害ラフィド藻Chattonella marinaおよびHeterosigma akashiwo遺伝子の検出法の検討を行った.プライマーおよびプロー ブ(TaqMan プローブ) は,C. marinaについてはmtDNA のcyt c 領域に,H. akashiwo についてはrRNA遺伝子のITS 領域に設定した.標的遺伝子の希釈系列を鋳型に用いた定量PCR の結果,高い増幅効率(C.marina:80.0 %; H. akashiwo:82.6 %)が確認され,さらにCt 値と遺伝子コピー数との間に高い正の相関(C. marina:R^2 = 0 . 991; H. akashiwo:R^2 = 0 . 998)が認められた.また,他種藻類(渦鞭毛藻,珪藻等)からDNeasy Plant Mini Kit(QIAGEN)を用いて抽出したDNA に対して非特異的増幅を示さなかった.さらに両種を対数増殖期から衰退期まで培養し,検鏡および定量PCR による細胞密度の定量を行った結果,両定量値間には有意(p<0.05)な正の相関(C. marina:R^2 =0.84;H. akashiwo:R^2 =0.96)が観察された.以上の結果から,DNA 抽出から定量PCR に至る本法の有用性が強く示唆された.
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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