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Molecular detection of Mycoplasma gallisepticum based on whole genome data
KURÁŽ, Jakub
This thesis reviews the basic knowlegde of a bacteria Mycoplasma gallisepticum and attempts to propose a functional detection protocol to distinguish M. gallisepticum from related species of the genus Mycoplasma. Real time PCR optimalization has refined the specificity and increased the efficiency of detection of selected genomic loci for M. gallisepticum. The functionality and accuracy of the method was verified in laboratory experiments by detecting the occurrence of M. gallisepticum in mixed Mycoplasma samples.
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Analysis of autenthicity of food products with fruit component
Prachárová, Adriana ; Mikulíková, Renata (referee) ; Márová, Ivana (advisor)
The aim of this thesis was to determine the authenticity of fruit food for infants using molecular and instrumental methods. In the experimental part, plant DNA isolations from fruit leaves (peaches, apricots, plums and apples) and bananas were performed. Further, DNA was isolated also from five commercial products, and from model mixtures that were prepared in terms of content identical to the commercial mixtures. The isolated DNA was characterized and verified by qPCR with plant DNA-specific ITS2 primers. Three triple primer pairs were selected, and their specificity was evaluated when performing multiplex PCR. This method makes it possible to detect more types of fruit in one reaction, reducing the economic and time requirements for detection. As none of the selected primer pairs were sufficiently specific for the apricot, the evidence from the plum and peach was further realized using duplex PCR. High resolution melting curve analysis was used for better DNA type recognition. Subsequently, agarose gel electrophoresis was performed to analyse the fragment lengths. Furthermore, experiments have been made to identify some specific phenolic substances in commercial and model fruit mixtures by HPLC. Since phenolic substances are degradable under unsuitable storage conditions, the presence of individual compounds was not detected by this method.
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Determination of selected red fruit species in plant-based food using multiplex PCR and instrumental methods
Vybíralová, Natálie ; Langová, Denisa (referee) ; Fialová, Lenka (advisor)
In many cases food is becoming the subject of adulteration, including fruit products that contain raspberries, strawberries and blueberries. This bachelor thesis is focused on the detection of strawberries, rapsberries and blueberries in model and commercial baby food products using multiplex PCR and HLPC. The theoretical part of this thesis is focused on composition of red fruits, their importance in human nutrition and especially about isolation of DNA from plant material. The aim of the experimental part of work was the analysis of selected commercial and model mixtures fruit purees containg raspeberries, strawberries and blueberries using instrumental and molecular biological methods. The results of these metods are compared. Commercial purees were bought in retail grocery shop. Model mixtures of these purees were prepared in our laboratory. DNA was isolated from fruit purees after and it’s amplifiability was comfirmed, it was successfully used in multiplex PCR to confirm the presence of raspberries, strawberries and blueberries in fruit purees. In the instrumental part, certain phenolic substences which are specific to red fruits were detected by HPLC in model and commercial mixtures.
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Detection of disease caused strain Xanthomonas phytopathogenic for tomato and pepper by multiplex PCR
RAGOZINA, Mariia
The subject of this work is the optimization of multiplex PCR for the detection of pathogenic bacteria of the genus Xanthomonas. Were successfully managed to optimized multiplex PCR reaction for simultaneous detection of Xanthomonas gardneri, Xanthomonas perforans, Xanthomonas euvesicatoria, and Xanthomonas vesicatoria using four primer pairs: Bs-Xg (Koenraadt et al., 2007), Bs-Xp (Koenraadt et al., 2007), Xe_330CG1,2 (Stehlíková a Beran, unpublished) and Xv_367CG1-1,9 (Stehlíková a Beran, unpublished). The selected primer pairs were also tested on a wide scale of other bacteria of Xanthomonas genus and non-Xanthomonas pathogens for tomatoes and peppers.
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Development of methods for genetic analysis of plant foods
Fialová, Lenka ; Brázda, Václav (referee) ; Doškař, Jiří (referee) ; Márová, Ivana (advisor)
Multiplex real-time PCR-HRM is an approach which has gained some attention in recent years. It has already found applications in clinical diagnostics and food authenticity and safety control. Compared to its corresponding singleplex PCR assays, an optimized multiplex PCR assay provides the same information in a fraction of time. First part of this work dealt with isolation of DNA from both fresh fruits and processed commercial products. Six different DNA isolation protocols were tested with fresh fruits – three silica column-based kits, two magnetic carrier-based kits and one conventional protocol. One method was chosen as the most suitable and was applied to DNA isolation from commercial products. These experiments also involved optimisation of the chosen method. The second part of this work was focused on the development of a triplex real-time PCR assay for simultaneous detection of blueberry, strawberry and raspberry, and its application on DNA isolated from commercial products. During DNA isolation, calcium chloride was shown to be a promising agent for removal of pectin from samples. In several samples, presence of raspberry DNA was confirmed by singleplex PCR. We found out that for accurate results of food analysis by this assay, further optimization of its parameters would be needed.
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Analysis of autenthicity of food products with fruit component
Prachárová, Adriana ; Mikulíková, Renata (referee) ; Márová, Ivana (advisor)
The aim of this thesis was to determine the authenticity of fruit food for infants using molecular and instrumental methods. In the experimental part, plant DNA isolations from fruit leaves (peaches, apricots, plums and apples) and bananas were performed. Further, DNA was isolated also from five commercial products, and from model mixtures that were prepared in terms of content identical to the commercial mixtures. The isolated DNA was characterized and verified by qPCR with plant DNA-specific ITS2 primers. Three triple primer pairs were selected, and their specificity was evaluated when performing multiplex PCR. This method makes it possible to detect more types of fruit in one reaction, reducing the economic and time requirements for detection. As none of the selected primer pairs were sufficiently specific for the apricot, the evidence from the plum and peach was further realized using duplex PCR. High resolution melting curve analysis was used for better DNA type recognition. Subsequently, agarose gel electrophoresis was performed to analyse the fragment lengths. Furthermore, experiments have been made to identify some specific phenolic substances in commercial and model fruit mixtures by HPLC. Since phenolic substances are degradable under unsuitable storage conditions, the presence of individual compounds was not detected by this method.
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Determination of selected red fruit species in plant-based food using multiplex PCR and instrumental methods
Vybíralová, Natálie ; Langová, Denisa (referee) ; Fialová, Lenka (advisor)
In many cases food is becoming the subject of adulteration, including fruit products that contain raspberries, strawberries and blueberries. This bachelor thesis is focused on the detection of strawberries, rapsberries and blueberries in model and commercial baby food products using multiplex PCR and HLPC. The theoretical part of this thesis is focused on composition of red fruits, their importance in human nutrition and especially about isolation of DNA from plant material. The aim of the experimental part of work was the analysis of selected commercial and model mixtures fruit purees containg raspeberries, strawberries and blueberries using instrumental and molecular biological methods. The results of these metods are compared. Commercial purees were bought in retail grocery shop. Model mixtures of these purees were prepared in our laboratory. DNA was isolated from fruit purees after and it’s amplifiability was comfirmed, it was successfully used in multiplex PCR to confirm the presence of raspberries, strawberries and blueberries in fruit purees. In the instrumental part, certain phenolic substences which are specific to red fruits were detected by HPLC in model and commercial mixtures.
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Mikrosatelitní panel pro srnčí zvěř a jeho využití
Podlipná, Eliška
As the name Microsatellite panel for roe deer and its use suggests, my bachelor thesis studies microsatellite markers for roe deer. The goal of this work was to construct microsatellite panel for roe deer and test it, while focusing on genetic variability of roe deer in ŠLP Křtiny and identifying the level of probability of railways and busy roads to be a barrier for the migration of roe deer and their gene flow. A total of 40 roe deer shot in the ŠLP Křtiny area in the 2017-2018 hunting season were sampled. Multiplex PCR was optimized for seven microsatellite markers. Statistical analyses showed presence of one population of roe deer in the ŠLP Křtiny area. Intraspecific differences were observed only on the level of individuals, they were absent on the level of potential groups. Moreover, the study showed that railways and busy roads present no barriers to migration of roe deer or their fluent gene flow. For further studies, I suggest to extend the number of markers for microsatellite panel and the area of roe deer sampling (for example different areas of the Czech Republic).
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Examination of microdeletion of chromosome Y in male infertile couples
VELÍŠKOVÁ, Taťána
In the last time the most popular topic has becouse infertility. The fertility of men is reduced every year. The cause may be wrong way of living, smoking, use of alcohol and drugs. In addition to can be chemical substances that enter the food and water. Next group of male infertility factors are the effects that lead to warning up the testicles. Optimal temperature is about 32 st. Celsius. People who are wearing tight clothes and have sitting job are proven to be fruitful. In the microdeletion chromosome Y, a long Y chromosome arm is examined on the AZF locus where the AZF, AZFb, AZFc regions are examined. Deletions in these positions are responsible for the failure of sperm formation and maturation and are portable to male offspring. Oligospermia or azoospermia may occur due to the absence of any of the AZFs. The aim of this bachelor thesis was to optimize the multiplex PCR method for the microdeletions in the azoospermic region of the chromosome Y (AZFa, AZFb, AZFc) and to evaluate the acquired data. In theoretical part are characterized diseases relacted to infertility man, for example azoospermic factor, Klinefelter's or Kallman syndrome. In practical part was examined from the scalp of the buccal mucosa 14 sample obtained from the man. All samples was negative, so every men were fruitful with suspicion of one infertile men.
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