Application of a Real-time PCR Assay to a Comprehensive Method of Monitoring Harmful Algae

概要

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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.