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Real-time PCR and it´s use in food processing
Tomanová, Barbora ; Pravečková, Martina (referee) ; Trachtová, Štěpánka (advisor)
Polymerase chain reaction (PCR) is a method abundantly used in molecular diagnostics. PCR in real time or quantitative PCR (qPCR) is one of its modifications and thanks to its advantages it finds still wider utilization nowadays. It finds its use in the food-processing industry too with relatively precise detection, identification, and qualification of both desirable and undesirable components in food, which often brings considerable difficulties and leads to an intensive development of this method. In the experimental part a DNA was isolated from dairy product Bio Via Natur drink for its further processing by means of PCR and gain more detailed information about a bacterial composition using real-time PCR and HRM analysis.
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Application of the method PCR-HRM analysis to identify bacteria in foods and food supplements
Šurková, Alice ; Illková, Kateřina (referee) ; Trachtová, Štěpánka (advisor)
Theoretical part of the thesis was focused on foods and food supplements containing microorganisms, especially bacteria. Furthermore, the thesis deals with methods for identification of the bacteria, primarily polymerase chain reaction (PCR). The thesis also includes real-time PCR and is specially focused on high resolution melting analysis (HRM). During the experimental part, the DNA sample was isolated from a chosen probiotics product using magnetic microparticles. The concentration of the DNA sample was determinate and DNA was subjected to PCR with subsequent detection PCR products by agarose gel electrophoresis. To the results specify HRM analysis was then performed.
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Optimization of DNA isolation form yogurt cultures and their detection by RT-PCR
Šurková, Alice ; Němcová, Andrea (referee) ; Brázda, Václav (advisor)
The thesis has optimized DNA isolation from pure yoghurt cultures and yoghurt products. The isolated DNA was than subjected to RT-PCR analysis. In the first part of the thesis, DNA isolation from pure yoghurt cultures using a commercial kit was evaluated as more effective than isolation by phenol extraction and magnetic microparticles. To assess the quality and quantity of DNA obtained the spectrophotometric determination of concentration and purity and qPCR were used. DNA of a total of ten pure yoghurt cultures in a quality suitable for PCR was obtained using the commercial kit. In the second part of the thesis, bacterial DNA was isolated from yoghurt products using the same commercial kit with a previous sample washing by lysation solution. DNA of six yoghurt products was isolated this way. Furthermore, two packages of homemade yoghurt were mad of each product, of which DNA was isolated in the same way. DNA obtained from yoghurts was subjected to RT-PCR using six pairs of primers (V3_F a V3_R, V6_F a V6_R, V1_F a V1_R, GroHRM_F a GroHRM_R, UPF a UPR, P1V1 a P2V1) and using the pure cultures DNA as a positive controls. The results confirmed the presence of cultures declared in each yoghurt and their ability to multiply after inoculation into a new medium (milk).
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Digital PCR development
Gaňová, Martina ; Lipov,, Jan (referee) ; Jansová,, Eva (referee) ; Neužil, Pavel (advisor)
Mikrotechnologické a nanotechnologické metody se v posledních letech ukázaly jako účinný nástroj pro analýzu deoxyribonukleové kyseliny (DNA). Různé techniky vyvinuté k amplifikaci nukleových kyselin (NK) byli publikovány v posledním desetiletí, včetně mikrofluidních systémů. Polymerázová řetězová reakce (PCR) je v molekulární biologii široce používána k amplifikaci cílové NK in vitro. Počet skupin pracujících s PCR po celém světě je obrovský vzhledem k důležitému sociálnímu a ekonomickému dopadu této techniky, například v oblasti lékařské diagnostiky, kriminalistiky, zpracování potravin nebo environmentálních studií. V současné době pandemie koronavirového onemocnění 2019 se prokázala důležitost vývoje přístupnějších technologií pro diagnostiku virových onemocnění. Disertační práce popisuje vývoj dvou verzí platforem PCR pro detekci NK, kapkovou kvantitativní PCR v reálném čase (qPCR) a digitální PCR (dPCR). Klíčové komponenty obou platforem byly vyrobeny pomocí mikrotechnologických postupů pro úpravu povrchů a litografickou výrobu umožňující vývoj hydrofobních krycích skel nebo křemíkových mikročipů. Výsledky práce demonstrují návrh, sestavení a testování včetně optimalizace obou platforem. Technologie PCR je tvořena softwarovou částí, ovládanou programem LabView a hardwarovou častí sestávající ze systému řízení teploty a zobrazovacího systém florescence. Kapková qPCR byla prováděna v 0.3 µL kapky směsi obsahující cílový gen napipetovaný v objemu 2 µL kapky minerálního oleje. Kapky byly pipetovány na hydrofobní krycí sklo, které bylo umístěno na termoelektrický chladič (TEC) pod fluorescenční mikroskop, aby se provedlo teplotní cyklování. Změny fluorescence během cyklů byly zachyceny fotonásobičem a sledovány osciloskopem. Výsledky testování popisují také schopnost multiplexování vyvinuté techniky. V práci je představená amplifikace tří syntetických genů s využitím interkalačního fluorescenčního barviva pro simultánní detekci a kvantifikaci na základě jednoho fluorescenčního kanálu. Kapková technologie qPCR byla zásadní platformou pro další vývoj platformy dPCR. Platforma dPCR používá křemíkový mikročip s disperzí vzorku do mikrojamek o celkovém počtu 26 448, každá s průměrem 50 µm a objemem 59 pL. Mikročip naplněn vzorkem byl pokryt minerálním olejem a krycím sklem modifikovaným polydimethylsiloxanem a Parylenem C. Systém ohřevu/chlazení teplotního cyklování s TEC byl podobný jako u kapkové platformy qPCR. Fluorescenční zobrazovací systém používal k zachycení fluorescenčních obrazů polovodičovou kameru na bázi CMOS. Vyvinutá dPCR byla testována pro aplikace ve výzkumu humánní medicíny. Testovací vzorky DNA byly část syntetického genu viru, izolovaná genomická DNA viru a izolovaná genomická DNA ženy. Disertační práce popisuje vývoj systému dPCR, který je součástí nové techniky dPCR, která je cenově dostupnější a snadno použitelná s jednoduchým dávkováním vzorků, což jsou nejčastější problémy, proč si zatím mezi laboratořemi nenašla velkou popularitu. dPCR mikročip na bázi křemíku zlepšil výkon systému díky velkému počtu mikrojamek. Využití technologie dPCR vyniká ve vysoké citlivosti, v nízkém poměru signálu k šumu, dosahované přesnosti, v nízkém detekčním limitu a schopnosti multiplexování.
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Interactions between microsporidial parasites and the host cladoceran Daphnia pulex in a simple environment of a forest pond
Krylová, Pavla ; Petrusek, Adam (advisor) ; Hyliš, Miroslav (referee)
Among the most common endoparasites who infected small crustacean Daphnia pulex include microsporidia. These intracellular parasites appear to look like a simple single- celled organisms, but their cell structure and lifecycle prove the opposite. Microsporidia are species-specific. Although they infected most organisms of the animal kingdom, they are not yet sufficiently understood. This theses is inderectly followed up to the studies of waterflea Daphnia longispina and microsporidia Berwaldia schaefernai from the dam reservoirs The aim was to analyze closer microsporidian infection on host Daphnia pulex in a forest pool with simple enviroment, which included monitoring time dynamics of Daphnia population and identification infection caused by microsporidia Berwaldia singularis and yet unknown microsporidia labor-marked "HVH". Laboratory work included determination of zooplankton and parasites, calculation of prevalence, laboratory experiments with transmission of microsporidian infection between healthy and infected flea culture or by isolated spores. Genetic analysis of aquatic invertebrates from the forest pool, especially larvae of mite and mosquitos, helped make closer microsporidian life cycle and hypothesis about secondary hosts, for the presence of pathogen DNA using specific DNA...
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The utilization of the uracil-DNA glycosylase in the elimination of carry-over contamination by products of the previous DNA amplifications
Zrnová, Adéla ; Martínková, Markéta (advisor) ; Prošková, Veronika (referee)
DNA amplification is an important tool in diagnosis of infectious diseases. The most commonly used method in laboratories is the polymerase chain reaction, which, does not meet the need for a fast and inexpensive method. It was therefore necessary to develop a method that would be sensitive, fast, specific and accessible, for example, in developing countries with limited access to instrumentation. It appears that LAMP could be this method. Unfortunately, the LAMP method encounters considerable false positivity, which must be prevented in order to be used in practice. The subject of the presented bachelor thesis is the introduction of a unique LAMP method to detect the presence of DNA Streptococcus pneumoniae and the study of the use of uracil- DNA glycosylase to prevent contamination of samples with DNA formed in previous amplification reactions. The LAMP method of DNA detection Streptococcus pneumoniae was successfully introduced, then the method was optimized even in the presence of deoxyuridine triphosphate. After determining the appropriate deoxyuridine triphosphate concentration for LAMP amplification, the resulting Streptococcus pneumoniae DNA amplification products were treated with uracil-DNA glycosylase. This procedure was shown to have the potential to prevent false positivity of other...
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Optimization of DNA isolation form yogurt cultures and their detection by RT-PCR
Šurková, Alice ; Němcová, Andrea (referee) ; Brázda, Václav (advisor)
The thesis has optimized DNA isolation from pure yoghurt cultures and yoghurt products. The isolated DNA was than subjected to RT-PCR analysis. In the first part of the thesis, DNA isolation from pure yoghurt cultures using a commercial kit was evaluated as more effective than isolation by phenol extraction and magnetic microparticles. To assess the quality and quantity of DNA obtained the spectrophotometric determination of concentration and purity and qPCR were used. DNA of a total of ten pure yoghurt cultures in a quality suitable for PCR was obtained using the commercial kit. In the second part of the thesis, bacterial DNA was isolated from yoghurt products using the same commercial kit with a previous sample washing by lysation solution. DNA of six yoghurt products was isolated this way. Furthermore, two packages of homemade yoghurt were mad of each product, of which DNA was isolated in the same way. DNA obtained from yoghurts was subjected to RT-PCR using six pairs of primers (V3_F a V3_R, V6_F a V6_R, V1_F a V1_R, GroHRM_F a GroHRM_R, UPF a UPR, P1V1 a P2V1) and using the pure cultures DNA as a positive controls. The results confirmed the presence of cultures declared in each yoghurt and their ability to multiply after inoculation into a new medium (milk).
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Interactions between microsporidial parasites and the host cladoceran Daphnia pulex in a simple environment of a forest pond
Krylová, Pavla ; Petrusek, Adam (advisor) ; Hyliš, Miroslav (referee)
Among the most common endoparasites who infected small crustacean Daphnia pulex include microsporidia. These intracellular parasites appear to look like a simple single- celled organisms, but their cell structure and lifecycle prove the opposite. Microsporidia are species-specific. Although they infected most organisms of the animal kingdom, they are not yet sufficiently understood. This theses is inderectly followed up to the studies of waterflea Daphnia longispina and microsporidia Berwaldia schaefernai from the dam reservoirs The aim was to analyze closer microsporidian infection on host Daphnia pulex in a forest pool with simple enviroment, which included monitoring time dynamics of Daphnia population and identification infection caused by microsporidia Berwaldia singularis and yet unknown microsporidia labor-marked "HVH". Laboratory work included determination of zooplankton and parasites, calculation of prevalence, laboratory experiments with transmission of microsporidian infection between healthy and infected flea culture or by isolated spores. Genetic analysis of aquatic invertebrates from the forest pool, especially larvae of mite and mosquitos, helped make closer microsporidian life cycle and hypothesis about secondary hosts, for the presence of pathogen DNA using specific DNA...
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Use of PCR in forensic genetic analysis.
Dvořáková, Lenka ; Šimková, Halina (advisor) ; Vaněk, Daniel (referee)
Polymerase Chain Reaction or PCR is molecular genetic method used to amplify the DNA fragment. Today it is one of the most popular and successful molecular genetic methods, which is used in many scientific and applied fields. PCR has many modifications derived from the classical scheme of reactions - for example multiplex PCR, inverse PCR, nested PCR, asymmetric PCR, and a lot more. The forensic genetic analysis is mainly used as a PCR amplification method of the studied loci for fragment analysis, as part of the sequencing and then quantified as a real-time PCR. The aim of this paper is to summarize the use of polymerase chain reaction in the forensic practice, and outline the methods in which PCR is used.
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Application of the method PCR-HRM analysis to identify bacteria in foods and food supplements
Šurková, Alice ; Illková, Kateřina (referee) ; Trachtová, Štěpánka (advisor)
Theoretical part of the thesis was focused on foods and food supplements containing microorganisms, especially bacteria. Furthermore, the thesis deals with methods for identification of the bacteria, primarily polymerase chain reaction (PCR). The thesis also includes real-time PCR and is specially focused on high resolution melting analysis (HRM). During the experimental part, the DNA sample was isolated from a chosen probiotics product using magnetic microparticles. The concentration of the DNA sample was determinate and DNA was subjected to PCR with subsequent detection PCR products by agarose gel electrophoresis. To the results specify HRM analysis was then performed.
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