Kochi University, Monobe, Nankoku, Kochi, Japan.
PLoS One. 2013 Mar 13;8(3):e57627. doi: 10.1371/journal.pone.0057627. Print 2013.
Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.
监测沿海水域的有害藻华（HAB）物种对于评估与 HAB 相关的环境影响非常重要。多种隐生种如有毒甲藻属物种的共现使得可靠的微观鉴定变得困难，因此通常需要使用分子工具。在这里，我们开发了一种新的 qPCR 方法，可以根据目标物种的实际基因数来计数隐生种的细胞。该 qPCR 检测方法针对来自日本的隐生种甲藻属 spp 的 LSU rDNA。首先，我们使用含有目标 rDNA 的线性化质粒构建了标准曲线。然后，我们通过使用 qPCR 检测方法从环境样品中分离的单个细胞中确定目标物种的每个细胞的 rDNA 拷贝数。通过在 DNA 提取前后将外源性质粒添加到样品裂解液的一部分中，计算 DNA 回收效率的差异，然后使用 qPCR 进行测定。然后，通过将样品中每个物种的总 rDNA 拷贝数除以每个细胞的 rDNA 拷贝数来计算每个物种的细胞数。为了测试我们的程序，我们使用含有无目标细胞但用几种甲藻属培养细胞接种的环境样品来确定 rDNA 的总数。qPCR 方法估计的数量非常接近添加的细胞总数。最后，通过 qPCR 方法对含有隐生种的环境样品中的目标物种的细胞数进行了计数，总数也非常接近显微镜细胞计数。我们开发了一种 qPCR 方法，可以准确地对环境中的每个隐生种进行计数。该方法有望成为监测沿海水域中各种作为隐生种出现的 HAB 物种的有力工具。
