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

Abstract

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.