Japan Fisheries Research and Education Agency, Fisheries Resources Institute, Fisheries Stock Assessment Center, 2-12-4 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan; Office of Industry-Academia-Government and Community Collaboration, Hiroshima University, 1-3-2 22 Kagamiyama, Higashi-Hiroshima City, Hiroshima 739-8511, Japan.
Japan Fisheries Research and Education Agency, Fisheries Resources Institute, Fisheries Stock Assessment Center, 2-12-4 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan; Department of Marine Systems, Tallinn University of Technology, Akadeemia tee 15A, 12618 Tallinn, Estonia.
Harmful Algae. 2021 Mar;103:102008. doi: 10.1016/j.hal.2021.102008. Epub 2021 Mar 15.
Recent increase of Harmful Algal Blooms (HAB) causes world-wide ecological, economical, and health issues, and more attention is paid to frequent coastal monitoring for the early detection of HAB species to prevent or reduce such impacts. Use of molecular tools in addition to traditional microscopy-based observation has become one of the promising methodologies for coastal monitoring. However, as ribosomal RNA (rRNA) genes are commonly targeted in molecular studies, variability in the rRNA gene copy number within and between species must be considered to provide quantitative information in quantitative PCR (qPCR), digital PCR (dPCR), and metabarcoding analyses. Currently, this information is only available for a limited number of species. The present study utilized a dPCR technology to quantify copy numbers of rRNA genes per single cell in 16 phytoplankton species, the majority of which are toxin-producers, using a newly developed universal primer set accompanied by a labeled probe with a fluorophore and a double-quencher. In silico PCR using the newly developed primers allowed the detection of taxa from 8 supergroups, demonstrating universality and broad coverage of the primer set. Chelex buffer was found to be suitable for DNA extraction to obtain DNA fragments with suitable size to avoid underestimation of the copy numbers. The study successfully demonstrated the first comparison of absolute quantification of 18S rRNA copy numbers per cell from 16 phytoplankton species by the dPCR technology.
近年来，有害藻华（HAB）的频繁出现导致了全球性的生态、经济和健康问题，因此人们更加关注对赤潮物种的频繁沿海监测，以便及早发现，预防或减少此类影响。除了传统的基于显微镜的观察外，分子工具的使用已成为沿海监测的一种很有前途的方法。然而，由于核糖体 RNA（rRNA）基因通常是分子研究的目标，因此必须考虑物种内和物种间 rRNA 基因拷贝数的变异性，以便在定量 PCR（qPCR），数字 PCR（dPCR）和代谢组学分析中提供定量信息。目前，这种信息仅可用于有限数量的物种。本研究利用 dPCR 技术，使用新开发的通用引物组和带有荧光染料和双猝灭剂的标记探针，对 16 种浮游植物物种（其中大多数是产毒物种）的每个单细胞的 rRNA 基因拷贝数进行定量。使用新开发的引物进行计算机 PCR 可以检测到 8 个超组的分类单元，证明了引物组的通用性和广泛的覆盖范围。发现 Chelex 缓冲液适合用于 DNA 提取，以获得大小合适的 DNA 片段，从而避免低估拷贝数。该研究成功地展示了首次通过 dPCR 技术对 16 种浮游植物物种的每个细胞的 18S rRNA 拷贝数进行绝对定量的比较。
