TY - THES A1 - Kanwal, Hafsa T1 - Detection of the Fish Pathogen Vagococcus salmoninarum via Quantitative real-time PCR for Aquaculture N2 - 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 Vagococcus salmoninarum, that is a causative agent of Vagococcosis. For this work, a primer and probe set specific to V. salmoninarum 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 V. salmoninarum being a Gram-positive bacteria, 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 Vagococcus salmoninarum in the PHOTO-SENS project. Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:hbz:1383-opus4-16222 ER - TY - THES A1 - Birkenbach, Gesa T1 - Quantitative real-time PCR Detection of Yersinia ruckeri for Application in Aquaculture N2 - To detect pathogen infections in aquaculture early and reduce their impact, a photonic biosensing chip is being developed in the PHOTO-SENS project, to enable fast and easy routine testing in aquaculture facilities. One target bacterial pathogen is Yersinia ruckeri, the causative agent of enteric redmouth disease. Detection is based on short DNA sequences (probes), that are complementary to the DNA of their target pathogen. In this work, a Yersinia ruckeri-specific probe was designed together with a set of qPCR primers. The probe and primers were tested in qPCR experiments, including quantifi- cation of extracted DNA, specificity and sensitivity testing, and detection of bacterial DNA from highly diluted samples to mimic the detection of environmental DNA. Three different standard series were created for the quantification of unknown samples. The influence of fish tissue on detection was tested, and the efficiency of Chelex-DNA ex- tractions were compared to peqGOLD Tissue DNA Mini Kit extractions. During quantification experiments with a first standard series (Y1P), the quantification output of most samples was 12–175 times higher than expected. The lower limit of detection was at 100 copies based on that standard, with an efficiency of 88%. The discrepancy was reduced with a gBlocks standard (limit of detection: 5 copies; effi- ciency: 92%). A CFU standard provided quantification results in reasonable agreement with expected CFU starting quantities (limit of detection: 1 CFU; efficiency: 106%). De- tection of diluted samples after filtration was possible. Chelex DNA extractions yielded higher amounts of detectable DNA compared to a DNA extraction kit. Less bacterial DNA was extracted in bacterial culture samples containing fish tissue than in samples that did not contain fish tissue. Higher-than-expected quantification outputs were attributed to flaws in the creation of a first standard series as well as detectable DNA from dead cells or clusters that were not accounted for in the expected starting quantities. The gBlocks standard was as- sumed to be most reliable for quantification of exact copy numbers, while the CFU standard produced results that could be accounted for in CFU counts. As these chal- lenges concerned the standard series’ and not the probe itself, it was concluded that the probe was suitable for application in the PHOTO-SENS project for detection of Yersinia ruckeri. KW - Quantitative real-time PCR (qPCR) KW - Yersinia ruckeri KW - DNA quantification KW - Hydrolysis probe KW - Aquaculture Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:hbz:1383-opus4-16361 ER -