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Influence of production environment on the occurrence of contaminating Penicillium in smear ripened cheese
Veselá, Vendula ; Brázda, Václav (referee) ; Vítová, Eva (advisor)
The topic of this thesis is the monitoring of the environment in a cheese production plant specializing in cheese ripened under a smear rind. The aim is to evaluate the occurrence of Penicillium mold as the main contaminant microorganism, depending on the sampling location and production time. Air samples were collected throughout the production process using the sedimentation method at 21 designated locations. The total number of microorganisms, selected groups of microorganisms, and the target microorganism (Penicillium mold) were monitored in the collected samples using cultivation (according to ČSN standards), microscopic, and molecular diagnostic techniques. The aim was to evaluate the occurrence of Penicillium citrinum, chrysogenum and commune species in dependence on the sampling location and production time. As part of the production screening, the broader microflora was first evaluated, followed by monitoring for contaminating molds. Using macroscopic and microscopic methods, 36 morphologically distinct colonies were selected. Microscopic analysis confirmed the presence of bacteria, yeasts and molds of the genera Penicillium, Cladosporium and Aspergillus. In order to identify the microorganisms present, an analysis using the polymerase chain reaction (PCR) method was performed, preceded by DNA isolation using the NucleoSpin Microbial DNA kit and phenol extraction. Based on real-time PCR results, the occurrence of contaminating Penicillium and Cladosporium molds was detected even in the early stages of production. Molds from these genera are found throughout the entire facility except for the single room that serves as a curd cooler before forming. Aspergillus mold, in the form of yellow mold with white mycelium on the edge of the colony, was detected only once in the drying room where cheese ripening occurs, and confirmed by microscopic observation. The genus Cladosporium was found in five morphologically distinct forms in the production environment. In addition, DNA was isolated from 11 morphologically distinct bacteria. Real-time PCR identified this DNA as belonging to coryneforms, with four of them belonging to the genus Brevibacterium. The genus Penicillium was represented by 13 morphologically distinct colonies, real-time PCR confirmed the presence of Penicillium commune and Penicillium chrysogenum. In addition to the mentioned molds, 11 morphologically distinct bacteria were detected, from which DNA was isolated. Real-time PCR identified this DNA as belonging to coryneforms, with 4 of them belonging to the genus Brevibacterium. The results of this work completely map the contamination of the production environment of the plant, and based on these results, methods for environmental sanitation were proposed to ensure the decontamination of the production process.
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Detection of pathogens in joint infections, including total endoprostheses using the BioFire method (Biomérieux)
ŠTĚTINOVÁ, Lucie
This bachelor thesis deals with the detection of pathogens in joint infections including total joint replacements by the BioFire method (Biomérieux, France). Compared to conventional methods, the Biofire Joint Infection Panel (BioMérieux, France) offers simultaneous detection of up to 39 pathogens and antimicrobial resistance markers in about one hour of time.This method is a multiplex PCR system for rapid and comprehensive testing. It is an innovative method that, thanks to its speed and comprehensiveness, will allow clinical microbiologists and physicians to make timely decisions on appropriate antibiotic therapy. Early de-escalation of this antibiotic therapy will also be enabled through the timely and targeted deployment of the appropriate ATB. The theoretical part of the thesis discusses the major infections of joints and joint replacements, introduces the reader to the most important causative agents of these infections, and a section is also devoted to microbiological diagnosis. The practical part describes the classical multiplex PCR, the FilmArray multiplex PCR system and with it the BioFire Joint Infection Panel, which has been used in the analysis of synovial fluid in patients with suspected septic arthritis or periprosthetic infection. Data from the analysis are also evaluated in the practical part. The data are from March 2021 to June 2022 and are presented in the practical part in the form of tables and graphs. The conclusion of the paper summarizes the relevance of the method for routine diagnosis and answers the stated research questions.
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Molecular genetic diagnostics of solitary fibrous tumors
ZÍTOVÁ, Lucie
The aim of this work is to provide an expert review of solitary fibrous tumors (SFT). In the practical part of the work, main aim is to create a comprehensive view of the molecular diagnosis of SFT. These tumors exhibit a broad spectrum of biological behavior from benign to malignant. The prognosis is generally uncertain. Promising marker of aggressive biological behavior is a mutation in the promoter region of the TERT gene, which causes the reactivation of telomerase reverse transcriptase. In this context, the key hypothesis of the work is the question of whether it is possible to determine the biological behavior of the tumor based on sequencing analysis of the promoter region of the TERT gene. A set of 33 patients of the University Hospital in Motol was analyzed and the data was statistically processed.
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PCR testing for selected diseases of bees
GREGOROVÁ, Sabina
Nosema ceranae is a species of microsporidia that negatively affects Apis mellifera. It is most commonly transmitted by coprophagy, or eating faeces when filling faecal sacs. It is a worldwide pathogen that is considered to be one of the causes of CCD (colony collapse disorder). The lesser known Crithidia mellificae belongs to the class Trypanosomatidae, which acts similarly like N. ceranae. Both of these pathogens target the guts of honey bees. As a result, infected bees weaken and the colony dies. The aim of this bachelor thesis was the practical examination of selected Apis mellifera samples from the beehive of the University of South Bohemia Faculty of Agriculture and Technology using the PCR method. In the theoretical part I describe Nosema ceranae and Crithidii mellificae and their effect on honey bees. Furthermore, the route of transmission of these pathogens and the possible treatment of infected honey bee colonies are mentioned. In the practical part, which I did in the laboratory at the Department of Plant Production of the Faculty of Agriculture and Technology, the procedure of DNA isolation, PCR and gel electrophoresis is described in detail. The evaluation of the results is also presented in this part.
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Optimization of isolation and PCR methods for the identification of CMS in onion
PETERKA, Václav
This bachelor thesis deals with the optimization of different methods of DNA isolation from different types of onion (Allium cepa L.) samples and the subsequent detection of the Ms locus and the detection of cytoplasmic male sterility (CMS). The different types of DNA extractions performed and their results were compared and evaluated. The best quality DNA was isolated from leaves using the CTAB-PVP. These test samples were also used for PCR for the detection of CMS and Ms locus using several different markers, which were compared with each other. The most suitable markers were then used in the PCR for the main samples. DNA isolation by CTAB-PVP was also used for further DNA isolation from the main samples of onion, which were two sterile lines and four fertile lines. The extracted DNA from these samples was first used to detect the Ms locus. Detection was performed using the marker AcSKP1. The samples were collected over several weeks. The Ms locus was detected in all samples (C1C6), regardless of the age of the plant. However, the Ms locus was detected in various forms of zygosity in samples from lines designated C2, C4 and C5. Furthermore, the DNA samples were used to detect CMS in plants and the type of CMS was determined. The markers cob and orfA501were used for this detection. Using these markers, it was found that the lines that were declared sterile (lines designated C1 and C4) were indeed sterile and had the CMS-T sterility type. The lines C2, C3 and C6 that were labelled as fertile were also detected as sterile samples. This means that these lines are not 100% fertile. In the line designated as C5, no sterility was detected in any of the samples.
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Development of a molecular methodology for the identification of insects used for human consumption
MÜLLEROVÁ, Veronika
Insect is often recognized as foodstuff of the future. An important requirement for insect use as a foodstuff is the development of an accurate and reliable method of insect detection. The goal of the bachelor thesis was to develop a molecular method that could be used for detecting the mealworm (Tenebrio molitor), an EU-approved species, in foodstuffs. The experimental part of the thesis included optimalization of mealworm DNA extraction. Specificity test of the method was performer on the superworm (Zophobas morio), which is not approved for consumption in the EU. DNA was extracted using 3 different methods. The optimal method achieving highest DNA yield and purity was DNA extraction with CTAB-PVP. Following DNA extraction, optimalization for PCR mix and cycle was peformed, which included optimalization of primer annealing. Results were visualised by gel electrophoresis. The optimized method is accurate, specific and appropriate for routine detection of the mealworm in foodstuffs.
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Classic vs. bulgarian yogurt - sensory quality vs. chemical and microbiological composition
Podloučková, Michaela ; Němcová, Andrea (referee) ; Vítová, Eva (advisor)
This thesis compares classic and Bulgarian yoghurt in terms of volatile content, sensory quality, and microbial profile. The volatiles were identified and semi-quantified by gas chromatography with mass detection in conjunction with solid phase microextraction, whereas classical culture followed by real-time polymerase chain reaction (PCR) was used to determine the microbial profile. Sensory analysis consisted of evaluation by graphic scales, profile and ordinal tests. In the experimental part, 3 samples were produced, a "classic" white and Bulgarian yoghurt made from commercial yoghurt cultures, and a traditional Bulgarian yoghurt imported directly from Bulgaria. Based on the PCR analysis performed, bacterial DNA was confirmed in all yogurts, only the traditional Bulgarian yogurt also contained yeast DNA. The Bulgarian traditional yoghurt thus differed in its detectable yeast taste, but above all in the qualitative and quantitative composition of the volatile substances. Of the 29 compounds identified, 20 were present in this yoghurt; esters were the main group, while ketones predominated in the other yoghurts. From a sensory point of view, the Bulgarian yoghurt made from commercial culture was the best evaluated, with a pleasant appearance, a pleasant smell and a pleasantly sour taste and an overall excellent sensory quality.
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Molecular identification of probiotic bacteria in milk products form commercial yoghurt cultures
Horňan, Samuel ; Fialová, Lenka (referee) ; Smetana, Jan (advisor)
Lactic acid bacteria are considered as an important group of bacteria with probiotic effects, which are being widely used in the food industry or pharmacology. Identification and characterization of important probiotic strains play an essential role in the validation of probiotic products for commercial purposes. Their identification using molecular-biology techniques (most commonly PCR method) is one of the standard tools in commercial operations and services. The aim of this bachelor thesis is a literature review of probiotics and probiotic strains as well as a summary of current knowledge about the use of molecular biology techniques for identification of these bacteria with probiotic properties in dairy products. The experimental part of this work verifies the presence of probiotic bacteria declared on selected commercial dairy products using the polymerase chain reaction (PCR) method.
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Use of molecular techniques to characterize yeasts of the genus Metschnikowia
Schneiderwindová, Nicole ; Brázda, Václav (referee) ; Němcová, Andrea (advisor)
This diploma thesis deals with the possibilities of implementation and use of molecular methods for the characterization of yeasts of the genus Metschnikowia and the application of methods in biotechnology or the food industry. The theoretical part focuses on a brief description of yeast, specially selected species that were used during the practical part of the work, the possibilities of their use, and especially on a detailed description of all molecular techniques used. The practical part focuses on the optimization of the molecular methods, namely the method of pulsed gel electrophoresis and the method of denaturing gradient gel electrophoresis. Initially, yeast was cultured under optimal conditions that are specific to this genus. Furthermore, their DNA was isolated using isolation techniques, which were subsequently processed using PFGE and PCR–DGGE methods. The DNA isolation procedure needed to be optimized the most. Several optimizations of the concentration of lysis enzymes, especially the lyticase enzyme, were performed. It was also necessary to determine the correct ratio of low-melting agarose and isolated DNA, which was essential for the correct consistency of the isolated DNA blocks and their further application in PFGE analysis. Finally, the PFGE method was optimized, which brought the correct distribution of chromosomes, and it was possible to describe the individual chromosomes according to their size according to the standard used CHEF of the yeast Hansenula wingei. To properly optimize the DGGE analysis process itself, it was first necessary to isolate the yeast DNA using a kit, then it was used as a template for the PCR reaction. The annealing temperature was also optimized for the individual groups of primers. The amplicons obtained by this reaction were separated by the DGGE method. This technique mainly required the optimization of basic parameters such as the range of the denaturation gradient or the total separation time. According to the measurement results, it can be determined that the process of yeast DNA isolation and their subsequent analysis using molecular methods of pulsed gel electrophoresis and denaturing gradient gel electrophoresis was successful. We were able to describe the genome and determine the number of chromosomes in all used yeast species of the genus Metschnikowia at least partially.
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The application of magnetic nano- and microparticles for the isolation of DNA from selected foods
Ráčková, Lucie ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
In thesis was verified micromethod for isolation of plant DNA from different vegetable (onion and broccoli) and plant food products in quality for application in polymerase chain reaction (PCR). The micromethod allows isolation DNA using magnetic particles from crude lysates of cells obtained by direct homogenization of plant tissues. Various methods of processing homogenates were compared. Homogenization was performed by lysis buffer containing cetyltrimethylammonium bromide (CTAB). The effect of the organic extraction agents was tested (chloroform-octanol and isopropanol). DNA was purified from homogenates by reversible adsorption on magnetic particles (four different types of magnetic particles were tested). The quality of isolated DNA was verified by UV spectrophotometry. The amplificabilty of DNA was tested by polymerase chain reaction (PCR). Specific primers for plant ribosomal DNA (rDNA) were used. PCR products of lenght 700 and 220 bp were detected by agarose gel electrophoresis. Differences in yield and quality of DNA were depended on the homogenate processing and magnetic particles used. The proposed procedure with two magnetic particles was tested for the isolation DNA from plan food products (spreads). DNA was amplified in PCR. Micromethod is suitable for DNA analysis of foods.
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