# Multiplex qPCR Assay for Simultaneous Detection of Three PST-Producing Dinoflagellates in the East China Sea off Southern Korea

## TL;DR

A new DNA-based test was developed to quickly detect three harmful algae species in seawater, helping prevent shellfish poisoning and improve coastal water monitoring.

## Contribution

A novel multiplex qPCR assay was developed for simultaneous detection of three PST-producing dinoflagellates.

## Key findings

- The assay successfully detected Alexandrium catenella and A. pacificum in field samples.
- The method showed strong linearity and high amplification efficiency with no cross-reactivity.
- Gymnodinium catenatum was not detected during the survey period.

## Abstract

Some microscopic algae in the ocean produce strong toxins that can accumulate in shellfish and cause serious food poisoning in humans. Detecting these harmful algae early is important to protect public health and marine ecosystems, but traditional identification methods are slow and often unreliable. In this study, we developed a DNA-based test that can quickly and accurately detect three toxin-producing algae species at the same time using a single analysis. This method was tested with seawater samples collected near southern Korea and successfully detected two harmful species that are commonly found in the region. The new test is fast, sensitive, and reliable, making it useful for routine monitoring of coastal waters. By improving early detection of toxic algae, this approach can help prevent shellfish poisoning events and support safer seafood production and marine management.

Paralytic shellfish toxins (PSTs) are produced by several toxic species of the dinoflagellate genera Alexandrium and Gymnodinium, and they pose significant threats to marine ecosystems and public health. Rapid and accurate detection of harmful algal blooms (HABs) is essential for effective management. In this study, we developed a multiplex quantitative real-time PCR (qPCR) assay targeting the 28S ribosomal DNA region to simultaneously detect three PST-producing dinoflagellates, Alexandrium catenella, A. pacificum, and Gymnodinium catenatum, in the East China Sea off southern Korea. Species-specific primers and hydrolysis probes labeled with distinct fluorophores were validated for simultaneous detection. The standard curves showed strong linearity (R2 > 0.99) and high amplification efficiencies (95.268–99.325%). No cross-reactivity was observed among the 20 non-target microalgal species. Field application of the assay using environmental DNA (eDNA) samples collected during spring successfully detected A. catenella and A. pacificum, whereas G. catenatum was not detected during the survey period. This multiplex qPCR assay provides a rapid and reliable molecular tool for early detection and spatial monitoring of potentially PST-producing dinoflagellates, supporting sustainable HAB management in East Asian coastal ecosystems.