The application of qPCR assays for the early detection of toxic Alexandrium in eastern australian waters
- Publication Type:
- Thesis
- Issue Date:
- 2019
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Harmful algal blooms that produce Paralytic Shellfish Toxins (PSTs) are prevalent and affect shellfish harvesting areas worldwide. PSTs have caused shellfish harvesting closures and product recalls, resulting in economic losses, as well as brand damage and damage to the wider economy including the tourism industry.
In Tasmania, it is known that four PST producing species co-occur, comprising ๐๐ญ๐ฆ๐น๐ข๐ฏ๐ฅ๐ณ๐ช๐ถ๐ฎ ๐ค๐ข๐ต๐ฆ๐ฏ๐ฆ๐ญ๐ญ๐ข, ๐. ๐ฑ๐ข๐ค๐ช๐ง๐ช๐ค๐ถ๐ฎ, ๐. ๐ข๐ถ๐ด๐ต๐ณ๐ข๐ญ๐ช๐ฆ๐ฏ๐ด๐ฆ and ๐๐บ๐ฎ๐ฏ๐ฐ๐ฅ๐ช๐ฏ๐ช๐ถ๐ฎ ๐ค๐ข๐ต๐ฆ๐ฏ๐ข๐ต๐ถ๐ฎ. In particular, of these, three species are morphologically almost identical, the species of the former ๐๐ญ๐ฆ๐น๐ข๐ฏ๐ฅ๐ณ๐ช๐ถ๐ฎ ๐ต๐ข๐ฎ๐ข๐ณ๐ฆ๐ฏ๐ด๐ฆ species complex (๐. ๐ค๐ข๐ต๐ฆ๐ฏ๐ฆ๐ญ๐ญ๐ข, ๐. ๐ฑ๐ข๐ค๐ช๐ง๐ช๐ค๐ถ๐ฎ, and ๐. ๐ข๐ถ๐ด๐ต๐ณ๐ข๐ญ๐ช๐ฆ๐ฏ๐ด๐ฆ), which cannot be differentiated using light microscopy. Therefore, phytoplankton monitoring using light microscopy and total PST in shellfish using High Performance Liquid Chromatography (HPLC) may not be sufficient to allow for an early warning with enough time to take appropriate shellfish harvesting management decisions.
In this thesis, quantitative Polymerase Chain Reaction (qPCR) assays are investigated as an in-field early warning system, as well as a tool for long-term risk assessment of PST-associated harmful algal blooms. A commercial on-farm pipeline based on the collection and filtration of water samples using a custom designed gravity filter, a cell lysis, and a qPCR assay based on ๐ด๐น๐ต๐4 was also developed and validated. QPCR assays based on ribosomal DNA (rDNA) โbarcodingโ regions and an assay based on a gene associated with PST biosynthesis (๐ด๐น๐ต๐4) were found to be generally specific, sensitive and efficient. The efficacy of an rDNA-based assay for cyst quantification was demonstrated, showing potential for its use as a long-term risk assessment tool for a new harvest area. However, qPCR assays based on rDNA gene regions were found to overestimate cell abundances. An analysis of rDNA copy number variation among strains of species of ๐๐ญ๐ฆ๐น๐ข๐ฏ๐ฅ๐ณ๐ช๐ถ๐ฎ showed a variation of up to 3-5 orders of magnitude within a species, and was correlated significantly with genome size, which also varied within a species. An analysis of the variation in genomic copies of ๐ด๐น๐ต๐4 genes showed variation as well, however this was of a lesser degree, of up to one order of magnitude. A positive correlation was found between ๐ด๐น๐ต๐4 copies per cell and the total PST produced per cell, showing that the dosage effect may contribute to the regulation of PST biosynthesis.
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