Novel Isothermal Nucleic Acid Amplification Assays for the Detection of Harmful Algal Species in Water and Shellfish Flesh.

Harmful algal blooms (HABs) are increasing in frequency and severity in both fresh- and salt-water environments. HABs deplete the water of oxygen and nutrients, and some species produce potent neurotoxins, which can accumulate in aquatic food-webs. Human exposure to HAB toxins through direct contact with contaminated waters, inhalation of aerosols or ingesting of aquatic foodstuffs can lead to a variety of severe ailments including paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), neurotoxic shellfish poisoning (NSP), amnesic shellfish poisoning (ASP), and azaspir acid shellfish poisoning (AZP). The economic impact in the US alone is estimated to be approximately $4.5Bn, annually. An early warning system for HAB species would contribute to an active management system to protect recreational waters, drinking water supplies and fisheries and shellfisheries from HAB-related losses. This could reduce the economic impact of HAB events, and protect human health. Conventional HAB surveillance practices employ multiple techniques including fluorometry, cytometry, microscopy and satellite imaging of ocean colour. Nucleic acid amplification tests (NATs) could complement these methods because they are highly specific for the target species and are particularly suited for automation. The latter enables analysis at the point of sample (POS) and in minimally resourced locations, and will be of particular importance as aquaculture expands into more remote and hazardous, offshore environments. Several isothermal NAT technologies have been developed over the past 30 years that show great promise for POS detection as the simplified thermal requirements and can function at low temperature, reducing instrumentation power and complexity. Here, several isothermal NAT assays have been developed to detect industry-relevant HABs, including Alexandrium minutum, Lingulodinium polyedra, Karenia brevis and Karenia mikimotoi. The novel methods detect rRNA and species-specific toxin biosynthesis mRNA transcripts in under 30 minutes. The novel assays have been tailored for use with a custom-built hand-held genetic sensor, named the Amplitron, to demonstrate the feasibility of rapid POS detection of HAB species.