A ubiquitous dinoflagellate, Alexandrium, produces paralytic shellfish toxin (PST), and its outbreaks have negative impacts on aquaculture, fisheries, human health, and the marine ecosystem. To minimize such damages, a routine monitoring program of toxic species must be implemented with a suitable analytical technique for their identification and quantification. However, the taxonomic identification and cell quantification of Alexandrium species based on their external morphology under a light microscope, or by using conventional molecular approaches have limited sensitivity and reproducibility. To address these challenges, we have developed an advanced protocol using droplet-digital PCR (ddPCR) for the discrimination and enumeration of three co-occurring Alexandrium species (A. affine, A. catenella, and A. pacificum) in environmental samples. Copies of species-specific internal transcribed spacer (ITS) per cell, which were calculated from environmental samples spiked with various numbers of culture cells, were used to estimate the abundance of species in the field samples. There were no significant differences in ITS copies estimated by the digital PCR assay between environmental samples from different localities, spiked artificially with a consistent number of cells from Alexandrium cultures. This sensitive assay was applied to determine the abundance and vertical distribution of those populations in the southern coastal waters of Korea. In spring, A. catenella was the dominant species, followed by the non-toxic A. affine in summers. A novel digital PCR assay can also be used to monitor other harmful marine protists that require high sample throughput and low detection limit with high accuracy and precision.

普遍存在的甲鞭毛藻亚历山大藻产生麻痹性贝类毒素（PST），其暴发对水产养殖，渔业，人类健康和海洋生态系统产生负面影响。为了最大程度地减少此类损害，必须使用适当的分析技术对有毒物种进行常规监测程序，以对其进行识别和定量。但是，基于亚历山大显微镜物种在光学显微镜下或通过使用常规分子方法的外部形态进行分类学鉴定和细胞定量分析的灵敏度和可重复性有限。为了解决这些挑战，我们已经开发了一种使用液滴数字PCR（ddPCR）的高级协议，用于区分和枚举三种同时出现的亚历山大种（A.仿射，链状亚历山大藻，和A. pacificum）环境样品英寸 每个细胞的物种特异性内部转录间隔子（ITS）副本是从加有不同数量培养细胞的环境样品中计算得出的，用于估算田间样品中物种的丰度。通过数字PCR分析估计的来自不同地区的环境样品之间的ITS拷贝没有显着差异，这些样品是由人工掺入了来自亚历山大文化的一致数量的细胞制成的。这项敏感的测定方法用于确定韩国南部沿海水域中这些种群的丰度和垂直分布。春季，A。catenella是主要物种，其次是夏季的无毒A.仿射。一种新颖的数字PCR分析法还可用于以高准确度和精确度监测其他需要高样品通量和低检测限的有害海洋生物。