Comparison of Two Molecular Methods, Quantitative Polymerase Chain Reaction and Sandwich Hybridization Assay, for Monitoring the Harmful Algal Bloom Species, Heterosigma akashiwo.

Abstract

Monitoring for harmful algal blooms (HABs) in aquatic environments is commonly aided by light microscopy, though molecular-based approaches can expedite species detection, cell quantification, and therefore early warnings for management responses. Two methods, quantitative polymerase chain reaction (qPCR) and sandwich hybridization assay (SHA), are increasingly used for HAB monitoring, but they differ in terms of protocols, genetic targets, equipment/supplies, and other considerations. This presents a challenge to end-users when selecting tool(s) to integrate within HAB surveillance programs. In response, we conducted a multi-year, side-by-side comparison study between qPCR and SHA relative to microscopy for monitoring the raphidophyte Heterosigma akashiwo, a species responsible for fish kills and impaired water quality worldwide. This paper summarizes key findings from a broad suite of side-by-side, laboratory and field tests of H. akashiwo cell quantification by qPCR and SHA. Assay ranges, detection limits, applicability to preserved samples, and physiological conditions (time of day, growth phase, nutrient levels) of cultured H. akashiwo revealed generally strong qPCR-SHA agreement, though qPCR had a wider dynamic range (without homogenate dilution) while SHA displayed a lower detection limit. Both assays yielded excellent agreement with microscopy during cell growth in the laboratory as well as during bloom development in the field. However, qPCR and SHA cell abundance data were less than microscopy during stationary-decline growth and under low nitrate, indicating reduced cellular nucleic acid during senescence and nutrient stress. Pragmatically, both qPCR and SHA are affordable, but qPCR solutions are typically more available commercially than SHA. Study results will be valuable to managers considering methodological options that suit their HAB monitoring needs.