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<p>Blooms of toxic <i>Karenia</i> species pose serious risks to fisheries, public health, and coastal ecosystems. Monitoring these harmful algal blooms (HABs) remains difficult because closely related, unarmored species are often morphologically indistinguishable. Here, we developed highly specific and sensitive TaqMan quantitative PCR (qPCR) assays for four ecologically important <i>Karenia</i> species (<i>K</i>. <i>longicanalis</i>, <i>K</i>. <i>mikimotoi</i>, <i>K</i>. <i>papilionacea</i>, and <i>K</i>. <i>selliformi</i>s)&#xa0;along the Chinese coast, with particular emphasis on the simultaneous identification of the morphologically similar, frequently co-occurring <i>K</i>. <i>mikimotoi</i> and <i>K</i>. <i>selliformis</i>. Species-specific primers and probes were designed to target the internal transcribed spacer (ITS) region for <i>K</i>. <i>longicanalis</i> and <i>K</i>. <i>selliformis</i>, the D1-D3 region of the large subunit ribosomal DNA (LSU rDNA) for <i>K</i>. <i>papilionacea</i>, whereas the assay for <i>K. mikimotoi</i> was established based on previously published primers and probe. All assays exhibited high specificity, with no cross-reactivity with non-target species. The assays were highly sensitive, with limits of detection as low as 2–3 gene copies μL<sup>−1</sup>, corresponding to 4–5 cells L<sup>−1</sup> in seawater. The assays were highly repeatable, with intra- and inter-assay coefficients of variation below 2%. We also optimized a duplex qPCR assay for the detection of <i>K</i>. <i>mikimotoi</i> and <i>K</i>. <i>selliformis</i>, achieving sensitivity comparable to that of the single-plex assays while substantially reducing processing time and cost. Field application of the qPCR assays to seawater samples collected during a <i>Karenia</i> bloom from the Beibu Gulf, Guangxi Province,&#xa0;South China Sea, confirmed the robustness and reliability of these assays for environmental monitoring. These species-specific molecular tools offer efficient and accurate methods for the early warning and detailed investigation of <i>Karenia</i> population dynamics during HAB events.</p>

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Establishment of TaqMan quantitative PCR assays for Karenia species along the Chinese coast

  • 期刊论文

  • 发表日期：

    2026年5月20日

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  • Kemeng Liu,
  • Yu Zhen,
  • Xiaoyu Chi,
  • Songhui Lu,
  • Jingyi Cen,
  • Jian Yuan,
  • Jianyan Wang

摘要

Blooms of toxic Karenia species pose serious risks to fisheries, public health, and coastal ecosystems. Monitoring these harmful algal blooms (HABs) remains difficult because closely related, unarmored species are often morphologically indistinguishable. Here, we developed highly specific and sensitive TaqMan quantitative PCR (qPCR) assays for four ecologically important Karenia species (K. longicanalis, K. mikimotoi, K. papilionacea, and K. selliformis) along the Chinese coast, with particular emphasis on the simultaneous identification of the morphologically similar, frequently co-occurring K. mikimotoi and K. selliformis. Species-specific primers and probes were designed to target the internal transcribed spacer (ITS) region for K. longicanalis and K. selliformis, the D1-D3 region of the large subunit ribosomal DNA (LSU rDNA) for K. papilionacea, whereas the assay for K. mikimotoi was established based on previously published primers and probe. All assays exhibited high specificity, with no cross-reactivity with non-target species. The assays were highly sensitive, with limits of detection as low as 2–3 gene copies μL−1, corresponding to 4–5 cells L−1 in seawater. The assays were highly repeatable, with intra- and inter-assay coefficients of variation below 2%. We also optimized a duplex qPCR assay for the detection of K. mikimotoi and K. selliformis, achieving sensitivity comparable to that of the single-plex assays while substantially reducing processing time and cost. Field application of the qPCR assays to seawater samples collected during a Karenia bloom from the Beibu Gulf, Guangxi Province, South China Sea, confirmed the robustness and reliability of these assays for environmental monitoring. These species-specific molecular tools offer efficient and accurate methods for the early warning and detailed investigation of Karenia population dynamics during HAB events.