Marine microbial biotoxins produced by certain phytoplankton species can accumulate at high levels in filter feeding bivalve shellfish. Monitoring programs in commercial shellfish aquaculture areas are essential to understand harmful algal blooms (HABs) and ultimately protect seafood consumers. Internationally and in Australia, the main harmful algal species that impact shellfish aquaculture are those that produce toxins associated with Paralytic Shellfish Poisoning (PSP), Diarrhetic Shellfish Poisoning (DSP), and Amnesic Shellfish Poisoning (ASP).
We present a novel approach for the simultaneous, rapid, and precise detection of Dinophysis species, paralytic shellfish toxins (PSTs), and certain target Pseudo-nitzschia species - causative agents of DSP, PSP, and ASP, respectively. Based on multiplex qPCR assay technology, we designed specific primers targeting the ITS ribosomal region to detect Dinophysis species and species belonging to the P. pseudodelicatissima complex Clade I; while for the detection of PSTs producing species, the qPCR assay targeted the functional sxtA gene, responsible for PSTs biosynthesis.
To evaluate the specificity, efficiency, and sensitivity of the multiplex qPCR assay, we tested a mock community composed of artificial genetic material of the three different targets. The multiplex assay successfully amplified all three targets, with efficiencies of 96.28% for Dinophysis species, 94.5% for P. pseudodelicatissima complex Clade I, and 91.38% for the sxtA gene.
The following steps for this research will evaluate environmental samples as targets. Further evaluation and comparison of this multiplex qPCR method, with current monitoring methods such as microscopy and biotoxin testing of shellfish flesh, has the potential to aid in a rapid and comprehensive understanding of HAB development, and act as an early warning monitoring system for food safety.