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.