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Identifier 000426962
Title Development of salmonella detection methods in food
Alternative Title Ανάπτυξη μεθόδων ανίχνευσης σαλμονέλας σε τρόφιμα
Author Κοντογιάννη, Γεωργία-Ιωάννα Π.
Thesis advisor Γκιζέλη, Ηλέκτρα
Abstract Outbreaks linked to Salmonella-contaminated products produce the need to develop simple, rapid and accurate detection methods, even if Salmonella is present in low amounts. In this study, we examined a novel strategy for the rapid detection and quantification of viable Salmonella by coupling a simple acoustic detection of biotinylated amplicons on neutravidin modified surface of a QCM-D sensor with loop-mediated isothermal amplification (LAMP). We first designed and optimized a LAMP assay targeting invA gene of Salmonella and secondly bcfD gene with biotinylated FIP primers. For low amounts of nucleic acids to be detected, 100nm POPC liposomes were used as a label in order to amplify the acoustic detection; this need for liposome binding in the LAMP products is the reason why we used loop forward and loop backward primers modified with cholesterol. 500 cells were used for LAMP amplification in the initial experiments (aiming to lower this threshold) and the reaction time had a range from 15 to 45 minutes, while control reactions took place to avoid the false negative and false positive results. In the case of invA gene the cholesterol probes were injected in the biosensor chamber after the injection of the biotinylated DNA and no signal was observed, so their presence was identified upon injection of POPC liposomes. For bcfD gene, two different approaches were used; in the first the injection of cholesterol probes in the biosensor chamber took place after the injection of the biotinylated DNA and in the second case, the cholesterol probes were used directly during the amplification (LAMP), so that the final product before the injection in the biosensor chamber contains biotin and cholesterol. In the first case no signal was observed after the addition of the POPC liposomes on the biosensor chamber regardless of the use or not of loop primers in the LAMP reaction. Nevertheless, in the case of the insertion of the cholesterol primers in the LAMP reaction, by-products were observed in the negative control reaction and similar signal shifts as in the positive reactions were recorded in QCM-D, leading to the conclusion that we cannot rely on those results. A neutralizers evaluation study was made for five plant occurring antimicrobial phenolic compounds (inhibitors) against two Salmonella strains: Salmonella Typhimurium and Salmonella Enteritidis. The five inhibitors, isolated as pure chemical compounds, were tested alone against the two heat treated strains in order to identify the minimum inhibitory concentration of those and then with the addition of five previously tested neutralizer recipes and twelve homemade. The heat treatment applied to the cells to check the recovery of the latter after the addition of an extra stress, except from the one because of the inhibitory matrices. The first screening of the five neutralizers was held against the pure chemical compounds and the second against raw materials in which it is contained each inhibitor. From the experiments we saw that Salmonella Enteritidis is more sensitive in the application of the thermal treatment and the majority of the experiments was performed with this strain. Even if after the first screening of the neutralizers against the pure chemical compounds we saw almost full recovery of the cells, on the second screening there was almost zero recovery in most of the trials. Those results indicate that the absence of recovery may be because the concentration of the neutralizers ingredients is not high enough to overcome the inhibitory effects since in all the toxicity and viability controls for the neutralizers there is full recovery.
Language English
Subject Ανίχνευση
Issue date 2019-11-29
Collection   Faculty/Department--Faculty of Sciences and Engineering--Department of Biology--Post-graduate theses
  Type of Work--Post-graduate theses
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