Department of Science and Environmental Studies, The Education University of Hong Kong, Tai Po, N.T., Hong Kong, China.
Department of Science and Environmental Studies, The Education University of Hong Kong, Tai Po, N.T., Hong Kong, China.
Mar Pollut Bull. 2017 Nov 30;124(2):890-896. doi: 10.1016/j.marpolbul.2016.12.043. Epub 2017 Jan 5.
Harmful algal blooms (HABs) have broken out frequently throughout the world in recent decades; they are caused by the rapid multiplication of algal cells in near-coastal waters polluted with nitrogen and phosphorus and greatly affect the quality of marine water and human health. Over the past several decades, climate change and increasing environmental degradation have provided favourable growth conditions for certain phytoplankton species. Therefore, it is essential to rapidly identify and enumerate harmful marine algae to control these species. In this study, quantitative PCR (qPCR) was used to detect four representative species of HABs that are widespread in the marine water of Hong Kong, namely, Alexandrium catenella, Pseudo-nitzschia spp., Karenia mikimotoi and Heterosigma akashiwo. We applied qPCR with the dye SYBR Green to detect Alexandrium spp. and Pseudo-nitzschia spp. and used TaqMan probe for the enumeration of Karenia mikimotoi and Heterosigma akashiwo. The total genomic DNA of these algae from Hong Kong marine water was extracted successfully using the CTAB method, and for each kind of alga, we constructed a ten-fold series of recombinant plasmid solutions containing certain gene fragments of 18S rDNA and ITS1-5.8S-ITS2 as standard samples. Ten-fold dilutions of the DNA of known numbers of the extracted algal cells were also used to create an additional standard curve. In this way, the relationship between the cell number and the related plasmid copy number was established. The qPCR assay displayed high sensitivity in monitoring marine water samples in which the low concentrations of harmful algae were not detected accurately by traditional methods. The results showed that the cell numbers of the four species were all in low abundance. For Alexandrium catenella, the cell abundances at 12 sites ranged from 3.8×10 to 4.3×10cellsL, while H. akashiwo, K. mikimotoi and Pseudo-nitzschia ranged from 1.1×10 to 1.3×10, from 23 to 6.5×10 and from 45 to 3.3×10cellsL, respectively. The concentrations of these algae were much lower than those observed during outbreaks of HABs in Hong Kong. These results may be useful for local aquaculture development and may provide effective suggestions and a theoretical basis for HAB monitoring and management.
有害藻华（HAB）在最近几十年在全球范围内频繁爆发；它们是由近海受氮、磷污染水域中藻类细胞的快速繁殖引起的，极大地影响了海水水质和人类健康。在过去几十年中，气候变化和环境恶化为某些浮游植物物种提供了有利的生长条件。因此，快速识别和计数有害海洋藻类以控制这些物种至关重要。在这项研究中，使用定量 PCR（qPCR）检测了在香港海洋水中广泛存在的四种代表性 HAB 物种，即链状亚历山大藻、拟菱形藻、米氏凯伦藻和赤潮异弯藻。我们应用 qPCR 结合 SYBR Green 染料检测链状亚历山大藻和拟菱形藻，并使用 TaqMan 探针对米氏凯伦藻和赤潮异弯藻进行计数。使用 CTAB 法成功提取了香港海洋水中这些藻类的总基因组 DNA，并且对于每种藻类，我们构建了包含 18S rDNA 和 ITS1-5.8S-ITS2 某些基因片段的十倍系列重组质粒溶液作为标准样品。还使用已知数量提取的藻类细胞的 DNA 进行十倍稀释，以创建附加的标准曲线。通过这种方式，建立了细胞数量与相关质粒拷贝数之间的关系。qPCR 检测法在监测海洋水样中表现出很高的灵敏度，而传统方法无法准确检测低浓度的有害藻类。结果表明，四种物种的细胞数量都很低。对于链状亚历山大藻，在 12 个位点的细胞丰度范围为 3.8×10 至 4.3×10cellsL，而赤潮异弯藻、米氏凯伦藻和拟菱形藻的细胞丰度范围分别为 1.1×10 至 1.3×10、23 至 6.5×10 和 45 至 3.3×10cellsL。这些藻类的浓度远低于香港 HAB 爆发期间观察到的浓度。这些结果可能对当地水产养殖的发展有用，并为 HAB 监测和管理提供有效的建议和理论依据。
