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Isolation of DNA from probitic products using solid carriers
Bonczek, Ondřej ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
Microbial DNA was isolated from lysed cells of Lactobacillus genus in probiotic products. Reversible adsorption DNA on the surface of carboxyl coated nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) magnetic particles and silicagel coated manganase Perovskite nanoparticles. DNA was adsorbed on the surface of the particles in the presence of 16 % poly(ethylenglycol) (PEG 6000) and 2 M sodium chloride (NaCl) concentrations. The adsorbed DNA was released from particles by low ionic strength TE buffer (pH= 8.0). The quality of isolated DNA was checked by spectrofotometric measurement and PCR amplification. DNA samples isolated using magnetic particles and phenol extraction method (control method) were PCR-ready. The DNA isolated from lysed cells of probiotic products was quantificated in real-time qPCR.
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Nucleic acids isolation for diagnostic purposes using polymeric carriers
Syslová, Ivona ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
The isolation of deoxyribonucleic acid (DNA) was studied in the diploma thesis by using three different methods: phenol extraction, salting with sodium chloride and magnetic separation with reversible adsorption of nucleic acids on different magnetic carriers. There were used five different properly functionalized carriers for the isolation of DNA: magnetic silicagel, P(HEMA-co-GMA) ox. I, P(HEMA-co-GMA) ox. II, Dynal DNA Direct and Perovskit 439. The reversible imobilization of DNA on the magnetic carrier was proceeded under the conditions of high concentration of NaCl and poly(ethyleneglycol) (PEG). There was induced the condensation of DNA by 2 M NaCl and PEG with molecular mass 6000 for binding of the DNA to the magnetic carriers and the final concentration of PEG in the separation mixture was 8 and 16 %. The aim was to gain the DNA of quality suitable for polymerase chain reaction (PCR). The DNA was isolated from the bacterial cultures of three probiotic strains, L. amylovorus CCM 4380T, L. zeae CCM 7069 T, L. plantarum CCM 7039T, which were cultivated in MRS medium. The DNA was also isolated from the fermented dairy products: Jihočeský zákys s ovocem jahoda (the fermented dairy product with the probiotic culture of Lactobacillus acidophilus, Bifidobacterium lactis and Streptococcus thermophilus), Revital active (the yogurt with inulin and the probiotic culture of Lactobacillus rhamnosus, Lactobacillus acidophilus and Bifidobacterium sp.) and Actimel višeň (the dairy product with the probiotic culture of Lactobacillus casei). When the PCR with the isolated DNA was passed off, the PCR products were detected by the gel electrophoresis with agarose. The success of the DNA isolation of the probiotic bacteria by phenol extraction, salting with NaCl and by magnetic separation, was verified by the PCR method. The method of magnetic separation using magnetic carriers was also verified for the isolation of DNA of quality suitable for PCR from the probiotic fermented dairy products.
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Identification and characterisation of selected properties of some strains of lactic acid bacteria
Sásková, Denisa ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
Nanotechnology is currently one of the fastest growing scientific disciplines. An interesting area of research is the biosynthesis of metal nanoparticles using microorganisms including lactic acid bacteria. In the first part this diploma thesis focuses on verification of identity of bacterial species Lactobacillus casei and Lactobacillus paracasei by genus- and species-specific PCR. In the next part of experimental work the capability of six Lactobacillus strains to produce silver nanoparticles is tested. Synthesis of nanoparticles was confirmed for all the strains depending on the amount of added AgNO3 and on time of cultivation. Differences between the strains were detected.
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Low power module with thermocamera
Horák, Daniel ; Najman, Jan (referee) ; Chalupa, Jan (advisor)
This bachelor thesis is focused on contactless temperature measuring with given thermographic camera and lossless image compression obtained by this camera. The theoretical part describes principles for better understanding of the issue and the practical part contains the solution itself, prepared for above mentioned thermographic camera.
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Izolation and identification of DNA from probiotic bacteria in complex matrices
Balogová, Petra ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
Nowadays, probiotic lactic acid bacteria (LAB) and bifidobacteria are plentifully exploited in food processing industry. LAB and bifidobacteria are important part of microflora of gastro intestinal tract (GIT). Probiotics (most often just lactobacilli and bifidobakteria) can be supplied to the GIT of the organism like food complements. Species identification is therefore very important. New methods of identification of LAB and bifidobacteria are based on analysis of DNA. Mostly exploited method is polymerase chain reaction (PCR). In my diploma work, genus and species specific PCRs were used for identification of different species of bacteria of genus Lactobacillus and Bifidobacterium in complex matrices of six food supplements (Zenflo, Linex Forte, Probian, Nutra Bona, GS lactobacillus Forte, Pangamin Bifi plus). Total DNA was isolated from crude lysates of cells present in tablets by magnetic particles coated by carboxyl groups . The preparation of cell lysates was optimalised. Different amounts of lysozyme (3 mg/ml, 10 mg/ml), time of incubation at laboratory temperature (1,5 hour, 3 hour) and time of incubation with SDS and proteinase K at 55 °C (1 hour, 3 hour, over all night) were tested. Isolated DNA was quantified and checked in PCR. Primers specific for genus Lactobacillus and Bifidobacterium and for species Lb. acidophilus, Lb. casei, Lb. rhamnosus and Lb. plantarum and B. animalis, B. bifidum, B. infantis, B. longum were used, respectively. All identified bacteria were in accord with the data declared by producer in 3 food supplements (Zenflo, Linex Forte and Pangamin Bifi Plus). The genus indentification was in accord with declaration of producer in other food products only (Probian, Nutra Bona, GS Laktobacily Forte).
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The use of magnetic microparticles for bacterial DNA isolation
Hrudíková, Radka ; Horák, Daniel (referee) ; Španová, Alena (advisor)
The aim of the work was testing of two types of magnetic mikrosheres functionalised with –COOH groups for the isolation of bacterial DNA. Isolation of DNA was carried out from crude lysates of cells prepared from pure culture of Lactobacillus paracassei RL-10 in the presence of binding buffer with 2 M NaCl and 16% PEG 6000. The influence of RNA degradation by enzyme RNase A on the amount of isolated DNA was investigated. It was estimated that RNA degradation affects the amount of DNA isolated. The amount of DNA depended on the type of microparticles. Higher amounts of DNA were isolated using particles with higher content of carboxyl groups. DNA applicable in PCR was isolated using both types of microsheres. In next part of the work, microparticles functionalised with –NH2 groups were used to DNA isolation using electrostatic forces. It was shown that buffer with lower pH is suitable for DNA adsorption onto magnetic microparticles.
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Software Design of Single Purpose Machine for Visual Inspection
Horák, Daniel ; Krejsa, Jiří (referee) ; Štarha, Pavel (advisor)
This master’s thesis deals with the fundamentals of machine vision application and its practical implementation. The research part is focused on the basic possibilities of image acquisition and image processing in different dimensions. The practical part describes the design of the dimension control algorithm using a 3D camera. This algorithm is then implemented in a single-purpose machine for optical dimension control.
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Photon-upconversion nanoparticles for single-molecule immunosensing of cancer biomarkers and bacteria
Farka, Z. ; Mickert, M. J. ; Hlaváček, Antonín ; Poláchová, V. ; Kostiv, Uliana ; Pastucha, M. ; Horák, Daniel ; Gorris, H. H. ; Skládal, P.
The recent progress in the field of immunoassays has been driven by introduction of various kinds of nanomaterials. In particular, photon-upconversion nanoparticles (UCNPs) proved to be excellent immunoassay labels due to their ability to emit light of shorter wavelengths under near-infrared excitation (anti-Stokes emission), which prevents autofluorescence, minimizes light scattering, and thus reduces the optical background interference. These unique photoluminescent properties allow counting of individual biomolecules labeled with UCNPs by conventional wide-field epiluminescence microscopy and enable the development of single-molecule (digital) immunoassays. We have introduced a novel label based on UCNPs conjugated with streptavidin via poly(ethylene glycol) and applied it in a digital upconversion-linked immunosorbent assay (ULISA) for the detection of a cancer biomarker prostate specific antigen (PSA). The digital readout based on counting of individual immunocomplexes improved the sensitivity 16× compared to conventional analog readout and allowed to reach a limit of detection (LOD) of 23 fg·mL−1 (800 aM). Human serum samples were successfully analyzed achieving an excellent correlation with electrochemiluminescence reference method. The conjugates of UCNPs with streptavidin are also suitable for the detection of pathogenic bacterium Melissococcus plutonius, the causative agent of honeybee disease European foulbrood. The ULISA assay provided an LOD of 340 CFU·mL−1 and successfully analyzed real samples of bees, larvae and bottom hive debris. Due to the high reliability and relatively simple detection scheme, the digital ULISA can pave the way for a new generation of digital immunoassays with a strong potential for commercialization.
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Low power module with thermocamera
Horák, Daniel ; Najman, Jan (referee) ; Chalupa, Jan (advisor)
This bachelor thesis is focused on contactless temperature measuring with given thermographic camera and lossless image compression obtained by this camera. The theoretical part describes principles for better understanding of the issue and the practical part contains the solution itself, prepared for above mentioned thermographic camera.
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