New real-time PCR assay for toxigenic Amphidoma languida

• Main Authors: Wietkamp, Stephan, Tillmann, Urban, Clarke, Dave, Toebe, Kerstin
• Format: Conference Object
• Language: unknown
• Published: 2018
• Subjects: Norway; Iceland
• Online Access: https://epic.awi.de/id/eprint/48263/; https://epic.awi.de/id/eprint/48263/1/ICHA_2018_Poster_SWietkamp.pdf; https://hdl.handle.net/10013/epic.4e76f99e-b6a0-4d74-add2-6ebf4ee18be4; https://hdl.handle.net/

Azaspiracids (AZA) are a group of lipophilic toxins, which are produced by a few species of the marine nanoplanktonic dinoflagellate genera Azadinium and Amphidoma (Amphidomataceae). Amphidomataceae were found to be globally distributed in coastal waters and new areas of occurrence are regularly discovered. The AZA toxins accumulate mainly in shellfish and - when consumed by humans - can lead to the so-called azaspiracid shellfish poisoning syndrome (AZP). Given this serious threat to seafood production and to deepen knowledge about the distribution and risk potential of AZA-producing algae, an appropriate detection method enabling a fast identification and quantification for these toxigenic species is needed. Traditional light microscopy is time-consuming, requires expertise and is getting rather difficult when it comes to the detection, identification and quantification of small-sized plankton. To overcome this challenges, quantitative real-time PCR (qPCR) assays are increasingly used as a molecular additive. Basically, when amplifying the extracted DNA and using DNA standards, the amplification threshold (CT) gives information about the number of target species in the sample. For two AZA-producing species, Azadinium spinosum and Azadinium poporum, quantitative PCR assays have already been developed and successfully applied in the field. Another AZA-producing species, Amphidoma languida, was discovered in 2012 in Irish coastal waters and discovered as a new species within the group of Amphidomataceae - in close relationship with Azadinium spp. All available strains from Ireland, Iceland, Norway, Denmark and Spain produce azaspiracids. Moreover, Am. languida from the Atlantic coast of southern Spain was responsible for AZA amounts in shellfish above the EU regulatory limit, emphasizing the need for further investigations. We thus developed a quantitative TaqMan PCR assay, amplifying 60bp of the D2 region (located on the LSU/28S) of the ribosomal DNA (rDNA) to detect toxic Am. languida. To confirm assay specificity in vitro, cross-reactivity tests with DNA of a variety of related organisms were performed. This included 12 different Am. languida strains as positive controls, Amphidoma parvula, 10 Azadinium species (each including different strains), as well as 10 further related dinoflagellate species (Alexandrium spp., Gymnodinium spp., Heterocapsa spp., Karlodinium sp., Prorocentrum spp. & Scripsiella sp.). The developed probe and primer set successfully detected only A. languida strains. Currently, we perform tests of the newly-designed assay on spiked field samples to test and optimize the quantification ability of the assay. With this assay, we provide a tool for the rapid and distinctive quantification of the toxic dinoflagellate Amphidoma languida to be used in monitoring programs and bio-geographic studies.