Development of isothermal nucleic acid amplification assay for toxicity studies on harmful algae

Supervisor: Jonathan McQuillan (National Oceanographic Centre, Southampton)
For half a century, the red tide-forming dinoflagellates Karenia brevis caused multiple episodes of toxic blooms contributing to severe impacts on ecosystems, human health, and economic activity for fishery, tourism and healthcare industries. K. Brevis produces a potent neurotoxin termed brevetoxin that may induce extensive loss in their algal competitors and acutely harm predators, thus increasing their prevalence and the frequency of harmful algal blooms in many parts of the world. Biosynthesis of brevetoxin is catalyzed by a set of polyketide synthase enzymes encoded via PKS genes. Experimental cultures of K. brevis were incubated under dimmed illumination to ensure cessation of photosynthetic functionality in algal cells. Dark incubation was applied over a period of 48 hours to limit K. brevis growth and evoke a physiological response which commonly involves brevetoxin production. A multiplexed format of an isothermal nucleic acid sequence-based amplification (NASBA) assay was designed to study the response of the K. brevis transcriptome in relation to light stress. Two molecular beacons of different fluorescence were employed to measure levels of rbcL and PKS in a single-tube amplification system. Diagnostic sensitivity and specificity of the assay were verified via on-chip electrophoresis of the NASBA products. Greatest levels in PKS gene expression occurred under reduced growth rates of K. brevis which tend to invest more carbon into defensive compounds for population survival and persistence demands. In parallel, Rubisco enzymes were insufficiently transcribed due to extreme down-regulation of carbon fixing gene (rbcL). Consequently, inhibition of carbon acquisition was leading to higher ambient concentrations of dissolved carbon and lower cultural pH values. In view of the ongoing increase in atmospheric CO2, toxicity of K. brevis blooms will most likely intensify on those far-reaching wide spectra of living organisms. The present study introduced for the first time a multiplex NASBA assay to monitor changes in transcriptomic profiles of rbcL and PKS encoding genes of K. brevis. As a result, the multiplex NASBA assay might be useful for high-throughput analysis of K.brevis transcriptional response and to elucidate underlying molecular mechanisms involved in brevetoxin biosynthesis.