Comparison of sandwich hybridization assay and quantitative PCR for the quantification of live and preserved cultures of Heterosigma akashiwo (Raphidophyceae)

Cameron Doll, Christopher R. Main, Colleen Bianco, Kathryn J. Coyne, Dianne I. Greenfield

Limnol. Oceanogr. Methods 12:232-245 (2014) | DOI: 10.4319/lom.2014.12.232

ABSTRACT: In this study, we directly compared two molecular techniques, sandwich hybridization assay (SHA) and quantitative PCR (qPCR), for quantifying laboratory cultures of the ichthyotoxic raphidophyte Heterosigma akashiwo. To maximize comparisons, all experiments entailed raising H. akashiwo in the laboratory, generating cellular homogenates then splitting them such that for each sample, the same homogenate was analyzed using both SHA and qPCR. To verify molecular data, all experiments included cell counts using light microscopy and a Sedgewick Rafter chamber. Results of standard curves for several geographically distinct strains using SHA were similar to prior research whereas others exhibited significantly different slopes, suggesting physiological differences between isolates. Data generated by qPCR showed a high degree of correlation with SHA responses. We also investigated the use of each method for quantifying H. akashiwo samples preserved with Lugol’s iodine solution. Neither SHA nor qPCR produced results that were significantly different from initial values for Lugol's preserved samples stored under refrigeration for up to 4 months. By comparison, samples stored at room temperature exhibited a highly significant decline in responses relative to controls after 4 months using both assays as well as a marked increase in ΔΔCt within 24 hours using qPCR followed by a decline and lack of signal after 1 month. To determine whether an environmental matrix would influence assay results, natural seawater samples were spiked with H. akashiwo culture. No significant differences between culture and spiked field samples were observed for either SHA or qPCR.