title: Quantitative PCR assay for detection and enumeration of ciguatera-causing dinoflagellate Gambierdiscus spp. (Gonyaulacales) in coastal areas of Japan.
abstract: In Japan, ciguatera fish poisoning (CFP) has been increasingly reported not only in subtropical areas but also in temperate areas in recent years, causing a serious threat to human health. Ciguatera fish poisoning is caused by the consumption of fish that have accumulated toxins produced by an epiphytic/benthic dinoflagellate, genus Gambierdiscus. Previous studies revealed the existence of five Gambierdiscus species/phylotypes in Japan: Gambierdiscus australes, Gambierdiscus scabrosus, Gambierdiscus sp. type 2, Gambierdiscus sp. type 3, and Gambierdiscus (Fukuyoa) cf. yasumotoi. Among these, G. australes, G. scabrosus, and Gambierdiscus sp. type 3 strains exhibited toxicities in mice, whereas Gambierdiscus sp. type 2 strains did not show any toxicity. Therefore, it is important to monitor the cell abundance and dynamics of these species/phylotypes to identify and characterize CFP outbreaks in Japan. Because it is difficult to differentiate these species/phylotypes by observation under a light microscope, development of a rapid and reliable detection and enumeration method is needed. In this study, a quantitative PCR assay was developed using a TaqMan probe that targets unique SSU rDNA sequences of four Japanese Gambierdiscus species/phylotypes and incorporates normalization with DNA recovery efficiency. First, we constructed standard curves with high linearity (R2=1.00) and high amplification efficiency (≥1.98) using linearized plasmids that contained SSU rDNA of the target species/phylotypes. The detection limits for all primer and probe sets were approximately 10 gene copies. Further, the mean number of SSU rDNA copies per cell of each species/phylotype was determined from single cells in culture and from those in environmental samples using the qPCR assay. Next, the number of cells of each species/phylotype in the mixed samples, which were spiked with cultured cells of the four species/phylotypes, was calculated by division of the total number of rDNA copies of each species/phylotype in each sample by the number of rDNA copies per cell. The numbers of cells of each species/phylotype quantified by qPCR assay were similar to the number of cells of each species/phylotype that were spiked. Finally, the cell densities of the target species/phylotypes were quantified using the qPCR assay in 30 environmental samples collected from Japanese coastal areas. Total cell densities of the four Gambierdiscus species/phylotypes quantified by qPCR assay were similar to those of Gambierdiscus spp. quantified by direct counting under a light microscope. The qPCR assay developed in this study is expected to be a powerful new tool for determining detailed distribution patterns and for monitoring the cell abundance and dynamics of each Japanese Gambierdiscus species/phylotype in the coastal areas of Japan.
authors.affiliation: LAQUES (Laboratory of Aquatic Environmental Science), Faculty of Agriculture, Kochi University, 200 Otsu, Monobe, Nankoku, Kochi, 783-8502, Japan; The United Graduate School of Agricultural Sciences, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566, Japan. Electronic address: aquariumuirauqa@gmail.com.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Nishimura
authors.affiliation: LAQUES (Laboratory of Aquatic Environmental Science), Faculty of Agriculture, Kochi University, 200 Otsu, Monobe, Nankoku, Kochi, 783-8502, Japan. Electronic address: hariganeya@hotmail.com.
authors.forename: Naohito
authors.initials: N
authors.lastname: Hariganeya
authors.affiliation: LAQUES (Laboratory of Aquatic Environmental Science), Faculty of Agriculture, Kochi University, 200 Otsu, Monobe, Nankoku, Kochi, 783-8502, Japan; The United Graduate School of Agricultural Sciences, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566, Japan. Electronic address: humer9@gmail.com.
authors.forename: Wittaya
authors.initials: W
authors.lastname: Tawong
authors.affiliation: LAQUES (Laboratory of Aquatic Environmental Science), Faculty of Agriculture, Kochi University, 200 Otsu, Monobe, Nankoku, Kochi, 783-8502, Japan. Electronic address: nufc-9@hotmail.co.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakanari
authors.affiliation: LAQUES (Laboratory of Aquatic Environmental Science), Faculty of Agriculture, Kochi University, 200 Otsu, Monobe, Nankoku, Kochi, 783-8502, Japan. Electronic address: yharuo@kochi-u.ac.jp.
authors.forename: Haruo
authors.initials: H
authors.lastname: Yamaguchi
authors.affiliation: LAQUES (Laboratory of Aquatic Environmental Science), Faculty of Agriculture, Kochi University, 200 Otsu, Monobe, Nankoku, Kochi, 783-8502, Japan. Electronic address: madachi@kochi-u.ac.jp.
authors.forename: Masao
authors.initials: M
authors.lastname: Adachi
mesh_terms: D004275:DNA, Ribosomal / Q000235:genetics; D004141:Dinoflagellida / Q000235:genetics* / Q000502:physiology; D004784:Environmental Monitoring / Q000379:methods*; D007564:Japan; D011156:Population Density; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results
chemical_list: D004275:DNA, Ribosomal
keywords: Ciguatera fish poisoning; Dinoflagellate; Gambierdiscus; Japan; Quantitative PCR assay