A digital PCR assay for the dabA gene involved in domoic acid biosynthesis by Pseudo-nitzschia spp

A digital PCR assay for the dabA gene involved in domoic acid biosynthesis by Pseudo-nitzschia spp

Weinstock C, Preston C, Brunson JK, Ussler W, Bowers H, Yamahara K, Doucette G, Ryan J, Allen A, Scholin C, Birch J

PMID: 41350003

Abstract

Successful harmful algal bloom (HAB) prediction and monitoring employs a variety of observational and research strategies. We add to the existing suite of tools for detecting toxigenic Pseudo-nitzschia diatoms by developing a novel digital PCR (dPCR) assay targeting a key gene of the domoic acid biosynthetic pathway, dabA. Sequence alignments and synthetic gene fragments of dabA genes from Pseudo-nitzschia australis, P. multiseries, P. multistriata, and P. seriata, along with the closely-related red-algal genes kabA and radA, were used to design a dPCR assay and assess its specificity. This dPCR assay was demonstrated to be specific for dabA and can quantify concentrations >1 copy µL in a reaction. The biogeography of published dabA genes suggests that the assay may be useful globally for target species. When screening a culture collection of Pseudo-nitzschia isolated from coastal California, the dPCR assay detected dabA genes from three of the eleven species tested. In samples collected during field campaigns in 2022 off Santa Barbara, CA and in 2023 in Monterey Bay, CA during toxic HAB events, Pseudo-nitzschia dabA genes were only detected in Monterey Bay samples. While the dPCR assay does not capture all dabA diversity, it is specific and provides a targeted means to assess the genetic potential of a toxic HAB event by dominant and potent toxin-producing Pseudo-nitzschia species in coastal California. Because dabA expression is a key indicator of DA production, using the assay to quantify gene transcription could speed the acquisition of data needed to forecast HABs before toxin is detected.