Universal rapid RNA-based quantification of toxigenic Alexandrium species (Dinophyceae) using quantitative recombinase polymerase amplification

Abstract

Harmful algal blooms caused by toxigenic Alexandrium species pose recurrent risks to coastal ecosystems and public health, yet current monitoring approaches rely on microscopy and laboratory-based toxin analysis with limited capacity for rapid, functional early warning. Here, we present a universal quantitative reverse-transcriptase recombinase polymerase amplification (qRT-RPA) assay targeting the sxtA4 transcript, an essential gene in saxitoxin biosynthesis. The RPA chemistry is isothermal with a low running temperature, making it suitable for portable, on-site testing. The assay was designed against a conserved sxtA4 region and validated using isolated sxtA4 amplicons from multiple Alexandrium species, synthetic DNA and RNA templates, and total RNA of Alexandrium minutum as a widespread reference species. The assay achieved uniform amplification kinetics across species, a limit of detection below 103 synthetic RNA copies and 0.1 ng total RNA per reaction for A. minutum, and a runtime of less than 15 minutes. The assay selectively detected sxtA4 transcripts from toxigenic Alexandrium strains and showed no cross-reactivity with non-target phytoplankton. A limit of detection that is relevant to early warning for Alexandrium species (under 20 cells per reaction) was retained in complex RNA matrices. Mock samples prepared by spiking cultured cells into natural seawater also demonstrated detection at field-relevant concentrations. These results establish qRT-RPA as a rapid, RNA-based, functionally informative molecular tool that provides a foundation for portable, early-warning monitoring of potentially toxigenic Alexandrium blooms.