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.
LAQUES (Laboratory of Aquatic Environmental Science), Faculty of Agriculture, Kochi University, 200 Otsu, Monobe, Nankoku, Kochi, 783-8502, Japan.
Harmful Algae. 2016 Feb;52:11-22. doi: 10.1016/j.hal.2015.11.018. Epub 2015 Dec 28.
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 (R=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.
在日本，近年来，赤潮鱼中毒（CFP）不仅在亚热带地区，而且在温带地区的报告也越来越多，对人类健康构成了严重威胁。赤潮鱼中毒是由食用积累了由附生/底栖甲藻属 Gambierdiscus 产生的毒素的鱼类引起的。先前的研究表明，在日本存在五种 Gambierdiscus 物种/类群：Gambierdiscus australes、Gambierdiscus scabrosus、Gambierdiscus sp. type 2、Gambierdiscus sp. type 3 和 Gambierdiscus（Fukuyoa）cf. yasumotoi。其中，G. australes、G. scabrosus 和 Gambierdiscus sp. type 3 菌株在小鼠中表现出毒性，而 Gambierdiscus sp. type 2 菌株则没有表现出任何毒性。因此，监测这些物种/类群的细胞丰度和动态对于识别和表征日本的 CFP 爆发非常重要。由于通过光显微镜观察很难区分这些物种/类群，因此需要开发一种快速可靠的检测和计数方法。在这项研究中，开发了一种使用 TaqMan 探针的定量 PCR 检测方法，该探针针对四个日本 Gambierdiscus 物种/类群的独特 SSU rDNA 序列，并结合 DNA 回收效率进行归一化。首先，我们使用包含目标物种/类群 SSU rDNA 的线性化质粒构建了具有高线性度（R=1.00）和高扩增效率（≥1.98）的标准曲线。所有引物和探针组的检测限约为 10 个基因拷贝。此外，使用 qPCR 检测方法从培养的单个细胞和环境样本中确定了每个物种/类群的每个细胞的 SSU rDNA 拷贝数。通过将每个样本中每个物种/类群的总 rDNA 拷贝数除以每个细胞的 rDNA 拷贝数，计算了混合样本中每个物种/类群的细胞数。通过 qPCR 检测方法定量的每个物种/类群的细胞数与每个物种/类群的细胞数相似。最后，使用 qPCR 检测方法对从日本沿海地区采集的 30 个环境样本中的目标物种/类群的细胞密度进行了定量。通过 qPCR 检测方法定量的四个 Gambierdiscus 物种/类群的总细胞密度与通过光显微镜直接计数的 Gambierdiscus spp. 相似。本研究中开发的 qPCR 检测方法有望成为一种强大的新工具，用于确定日本沿海地区每个 Gambierdiscus 物种/类群的详细分布模式，并监测其细胞丰度和动态。
