Development and Laboratory Validation of a Real-Time Quantitative PCR Assay for Rapid Detection and Quantification of Heterocapsa bohaiensis

Heterocapsa bohaiensis is an emerging harmful dinoflagellate increasingly reported from coastal regions of the Pacific. However, an available molecular assay offering rapid and sensitive detection is still lacking. This study developed a SYBR Green real-time quantitative PCR (qPCR) assay for the identification and quantification of H. bohaiensis. Species-specific primers (F: 5′-CCATCGAACCAGAACTCCGT-3′; R: 5′-AGTGTAGTGCACCGCATGTC-3′) were designed and the assay was optimized and evaluated using laboratory cultures for specificity, sensitivity, and quantitative performance. Primer screening and melt-curve analysis confirmed that the selected primer pair produced a single, specific amplification peak for H. bohaiensis, with no cross-reactivity observed in non-target species (Chlorella pyrenoidosa, Phaeocystis globosa, Skeletonema costatum, Alexandrium tamarense) or mixed algal communities. The standard curve displayed strong linearity (R2 = 0.9868) and a high amplification efficiency (102.5%). The limit of detection (LOD) was approximately 2–3 cells per reaction, as determined from 24 replicates of 5-cell equivalents and verified at ~2.7-cell equivalents. This sensitivity was comparable to or exceeded that reported for assays targeting other HABs forming dinoflagellates. Quantitative results derived from the qPCR assay closely matched microscopic cell counts, with a relative error of 10.79%, falling within the acceptable threshold for phytoplankton surveys. In summary, this study established and validates a species-specific qPCR assay for H. bohaiensis under controlled laboratory conditions. The method shows strong potential for incorporation into HAB monitoring programs, early-warning systems, and future ecological investigations of this emerging species.