PRIMER DESIGN & QPCR OPTIMISATION FOR DETECTING GEOSMIN-PRODUCING BACTERIA IN RAS

Abstract

“Off-flavour” compounds, such as geosmin, are responsible for the tainting of fish fillets and cause profit losses in closed systems. Geosmin is an earthy-smelling compound produced by certain bacteria and fungus and one of the more prevalent and problematic “off-flavour” compounds in recirculating aquaculture systems (RAS). This study aims to accurately quantify geosmin-producing bacteria to assess the optimal removal strategy of off-flavours from Atlantic salmon (Salmo salar) grown in brackish-water RAS, purging or in-combination with electrolysis. A primer was designed based on conserved regions of bacterial geosmin synthase sequences identified through multiple sequence alignments and in silico specificity analysis. This study developed and validated a qPCR protocol for the geosmin synthase gene using temperature and concentration gradients for optimisation. A synthetic DNA template was used to generate standard curves across two dilution series, achieving R2 values of 0.997 and 0.982 with amplification efficiencies of 102% and ~83%, respectively. Specific amplification was confirmed by melt curve analysis. Expression of geosmin synthase in gill and skin samples were measured across time points in both systems and were normalised to validated reference genes as controls for template variation. The established qPCR protocol and understanding of the geosmin synthase gene provides a reliable framework for quantifying geosmin synthase expression in environmental samples, supporting future evaluation of geosmin mitigation strategies in RAS.