the 22nd Pacific−Asian Marginal Seas meeting (PAMS)

With the accelerating impact of climate change, Korean coastal waters are becoming increasingly subtropical, raising the likelihood of harmful algal bloom (HAB)-forming species. Early detection of HAB species is essential for effective monitoring and management. Among them, the genus Dinophysis, which causes Diarrhetic Shellfish Poisoning (DSP), is difficult to culture under laboratory conditions, posing challenges for the development of accurate quantitative standards. To overcome this limitation, this study used artificially synthesized oligonucleotides as a substitute for Dinophysis DNA to construct a qPCR standard curve. Species-specific primers targeting the LSU rDNA region were designed, and the performance of the synthetic oligo-based standard curve was evaluated by comparison with Dinophysis genomic DNA. The spike experiment was conducted to improve the accuracy of cell density quantification in environmental samples. The sensitivity and specificity of the developed detection method were further evaluated using EvaGreen-based qPCR assays with field samples collected from Jinhae Bay and Jeju coastal waters in 2022 and 2023. As a result, in-silico analysis showed that the primer-binding regions were highly conserved among 14 Dinophysis species and matched 30 Dinophysis sequences in NCBI GenBank. Specificity tests further confirmed positive amplification only in Dinophysis acuminata among 14 dinoflagellate species, 1 diatom species and 1 raphidophyte species. The synthetic oligonucleotide-based standard curve showed high reliability (R² = 0.999) and yielded quantification results comparable to those obtained using Dinophysis genomic DNA. In addition, the spike experiment improved the accuracy of cell density estimation, and EvaGreen-based qPCR assays successfully detected Dinophysis in field samples, including low-density samples that were difficult to detect by microscopy. This approach enables effective analysis of environmental samples and demonstrates the potential of DNA sequence-based detection strategies for other non-culturable harmful microalgae. The method will contribute to improving HAB monitoring efforts in response to the ecological changes driven by climate change.
 

Synthetic olignucleotide,Dinophysis,Harmful Algal Blooms (HABs),eDNA