Journal of Applied Phycology

, Volume 30, Issue 4, pp 2425–2434 | Cite as

Critical comparison of molecular methods for detection and enumeration of the harmful algal species, Heterosigma akashiwo, in environmental water samples

  • Christopher R. Main
  • Dianne I. Greenfield
  • Cameron Doll
  • Yanfei Wang
  • Edward B. Whereat
  • Rebecca Mortensen
  • D. Tye Pettay
  • Kathryn J. CoyneEmail author


Molecular methods such as quantitative real-time PCR (qPCR) and sandwich hybridization assay (SHA) enable a more rapid and specific enumeration of harmful algal species compared to microscopic cell counts. Integrating these methods into routine monitoring and management strategies, however, has been slow. While comparisons to microscopy have been made, direct comparisons between molecular methods using environmental samples are sparse. Here, we directly compare qPCR to SHA for enumerating the harmful algal species, Heterosigma akashiwo, in environmental samples collected in Delaware’s inland bays. To ensure comparability, a single cellular homogenate was generated from field samples and split for analysis by qPCR and SHA. Results show a significant correlation between qPCR and SHA when Heterosigma is above bloom levels (1 × 105 cells L−1), but not during non-bloom conditions. qPCR and SHA were also more highly correlated when samples were collected at lower temperatures (< 25 °C) and/or with high levels of chlorophyll a (greater than or equal to 30 μg L−1), independent of Heterosigma cell concentration. There was no evidence of cross-reactivity in primers and probes for H. akashiwo during blooms of the closely related species, Chattonella subsalsa. However, qPCR to SHA ratios were elevated during blooms of other phytoplankton species, suggesting suppression of the SHA signal or enhancement of qPCR. Results of this study may have significant implications for research, where precise evaluations of cell numbers are often required. However, precise cell counts at non-bloom levels may not be as critical to management, suggesting either technique could be incorporated into rapid and effective decision making.


Heterosigma akashiwo qPCR Sandwich hybridization assay Harmful algal blooms Delaware inland bays Raphidophyte 



This project was funded by Delaware Sea Grant (grant R/HCE-4 to Coyne) and the National Oceanographic and Atmospheric Administration (NOAA) National Centers for Sponsored Coastal Ocean Research (award #NA10NOS4780141 to Greenfield and Coyne). This represents contribution numbers 210 from the NOAA Monitoring and Event Response for Harmful Algal Blooms (MERHAB) Research Program, 1860 from the Belle W. Baruch Institute, University of South Carolina, and 785 from the Marine Resources Research Institute, South Carolina Department of Natural Resources.

Supplementary material

10811_2018_1444_MOESM1_ESM.docx (22 kb)
Table S1 (DOCX 21 kb)
10811_2018_1444_MOESM2_ESM.pdf (13 kb)
Fig. S1 Microscopic identification and enumeration of potentially toxic or bloom forming species performed by the UD Citizen Monitoring Program. Cell counts of > 1.0 × 106 cells L−1 were based on average number of cells in at least 10 fields of vision. Limit of detection is 1.3 × 104 cell L-1 (PDF 12 kb)


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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Christopher R. Main
    • 1
    • 2
  • Dianne I. Greenfield
    • 3
    • 4
    • 5
  • Cameron Doll
    • 4
  • Yanfei Wang
    • 2
  • Edward B. Whereat
    • 2
  • Rebecca Mortensen
    • 3
  • D. Tye Pettay
    • 2
  • Kathryn J. Coyne
    • 2
    Email author
  1. 1.Delaware Department of Natural Resources and Environmental ControlDoverUSA
  2. 2.College of Earth, Ocean, and EnvironmentUniversity of DelawareLewesUSA
  3. 3.Belle W. Baruch Institute of Marine and Coastal SciencesUniversity of South CarolinaCharlestonUSA
  4. 4.South Carolina Department of Natural ResourcesMarine Resources Research InstituteCharlestonUSA
  5. 5.Advanced Science Research Center at the Graduate SchoolCity University of New YorkNew YorkUSA

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