Revealing species diversity in Pseudo-nitzschia blooms in Scotland and Aotearoa (New Zealand) by combining recombinase polymerase amplification and nanopore-sequencing

The planktonic marine diatom genus Pseudo-nitzschia is synonymous with the neurotoxin domoic acid (DA), which causes the syndrome amnesic shellfish poisoning (ASP). DA can bioaccumulate in shellfish during blooms of specific, toxin-producing Pseudo-nitzschia, harming wildlife and human health. Phenotypic discrimination of variants requires electron microscopic observation of the frustule, a process too time-consuming for routine monitoring, which invariably uses light microscopy. This process is less specific and has poor correlation with accumulation of DA in shellfish. The assay outlined here targets the ribosomal DNA gene, providing species resolution of Pseudo-nitzschia. It combines recombinase polymerase amplification (RPA) with nanopore sequencing and is effective on almost all known Pseudo-nitzschia species. It provides preliminary, real-time indication of environmental species composition, and further bioinformatic processing confirms results. Validation experiments correctly identified 13 reference cultures, with sufficient sensitivity (2500 cells l-1). Two environmental case studies highlighted efficacy on complex natural Pseudo-nitzschia blooms, including detection of low abundance species. The first study consisted of 37 samples collected from Scottish waters between May 2022 and August 2023, which identified 13 species. The second consisted of 10 samples from Aotearoa (New Zealand) collected February 2025, which found 17 species. Both studies identified species previously undetected in the regions. Statistical analysis of complementary toxin data from Scotland, although limited, highlighted that relative abundance of toxic species was a better predictor of toxic events than genus level cell counts. The RPA assay was also adapted to a lateral flow dipstick, providing a rapid screening tool for toxic Pseudo-nitzschia seriata and Pseudo-nitzschia australis.