Detection of the Fish Pathogen Aeromonas salmonicida via Quantitative real-time PCR for Aquaculture
PHOTO-SENS is a project that aims to develop photonic biosensing chips as a means of allowing onset detection of pathogenic infections in aquaculture. These chips are meant to be a way for quick and simple routine testing that would enable aquaculture facilities to recognize potential outbreaks and minimize their effects. The chips detect target bacteria using short DNA sequences known as probes which are complementary to target DNA. One of the fish pathogens under consideration for the course of this project is Aeromonas salmonicida, that is a causative agent of Furunculosis.
For this work, a primer and probe set specific to A. salmonicida was designed. These primers and probes were then tested via qPCR for their selectivity and sensitivity and most importantly, quantification of DNA in samples using two different standard dilution series. Due to A. salmonicida being a Gram-negative bacterium, three methods of DNA extraction were used and compared for maximum efficiency. Moreover, experiments were also conducted to mimic capture of eDNA (environmental DNA) from highly diluted water samples.
Upon comparing the three DNA extraction methods, the improved 5% Chelex protocol that included rapid agitation via glass beads, yielded the highest amount of DNA that was closest to expected SQ. The detection of all samples, including very low copy number, was possible via qPCR. The first standard series which was the gBlocks standard series had the lowest detection limit of 1 copy with an efficiency of 105.8% and the second standard series that is the CFU standard series had a detection limit of 10 copies and had an efficiency of 104.2%. The filtration experiment also showed detection in diluted, filtered samples.
Since detection using the designed probe was successful, the probes are therefore considered suitable for detection of Aeromonas salmonicida in the PHOTO-SENS project.