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Molecular genetic analysis of rare ocular disorders in Roma population from the Czech Republic
Rysková, Natálie ; Lišková, Petra (advisor) ; Klímová, Aneta (referee)
The Roma are the largest and most widespread transnational ethnic community. The Czech Republic estimates their 2% representation in its population. Due to the high level of endogamy, the spectrum and frequency of hereditary diseases in the Roma population differ from the majority population. Hereditary eye diseases are one of the most common causes of blindness in younger adults and thus represent a real socio-economic burden. The aim of the thesis was to perform molecular genetic analysis in individuals of Roma origin suffering from hereditary diseases affecting vision, including dual impairments and syndromes, and to determinate the frequency of the detected pathogenic variants in this population. Molecular genetic analysis of 17 families was performed using direct and whole exome sequencing. Within the framework of an international collaboration, the degree of their mutual kinship was calculated using the PLINK program. The frequency of selected variants was determined in a control dataset comprising 156 Roma exomes and genomes. The spectrum of analyzed diseases included various retinal dystrophies, primary congenital glaucoma, Usher syndrome, neuronal ceroid lipofuscinosis, Noonan syndrome, nanophthalmos and congenital cataract, facial dysmorphism and neuropathy. The results of the thesis...
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Kryptosporidie a kryptosporidióza chovaných a zdivočelých nutrií
LIMPOUCHOVÁ, Zlata
Cryptosporidium spp., common parasite of vertebrates, remain poorly studied in wildlife. This study describes the novel Cryptosporidium species adapted to nutria (Myocastor coypus). A total of 247 faecal samples of feral and farmed nutrias were collected from locations in the Czech Republic and Slovakia and examined for Cryptosporidium spp. oocysts and specific DNA at the SSU, actin, HSP70, and gp60 loci.
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Molekulární diagnostika fungálních patogenů z klinických vzorků
CIMICKÁ, Jana
This study is focused on optimization and use of molecular methods in mycological diagnostics. The patient's life often depends on early diagnosis, which current culture methods cannot provide. The solution is based on pan-fungal primers of ITS region and next generation sequencing (NGS). These methods were used for fungal detection from FFPE tissue samples from patients with ulcerative colitis (UC), Crohn disease (CD) and 10 controls without chronic inflammatory changes in the intestine. Results of this study have three levels. Firstly, the process of optimization highlights the importance of sterile DNA isolation in a laminar box which can prevent contamination by Malassezia spp. Secondly, raw data showed balanced rate of Ascomycota and Basidiomycota in CD samples similar to negative controls, while UC samples indicated higher representation of Ascomycota than other groups. Thirdly, detailed data showed fungal genera Malassezia, Cladosporium and Toninia in all groups, while genus Candida was found only in UC samples and genera Engyodontium and Ramularia were found in CD samples. In conclusion, mycobiome plays a role in inflammatory bowel diseases; however, its compartments are still questionable. The optimization of pan-fungal nested PCR and NGS helped with introduction of mycological diagnostics from FFPE samples to Bioptická laboratoř s.r.o. and is used for differential diagnostics.
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Molecular and serologic diagnosis of infections caused by Trichobilharzia
Vaščiková, Michaela ; Horák, Petr (advisor) ; Ditrich, Oleg (referee)
Cercariae of the genus Trichobilharzia can penetrate not only the skin of definitive hosts (ducks), but they are also able to penetrate the skin of accidental hosts (mammals). As a result of the penetration, the inflammatory response known as cercarial dermatitis appears. The goal of our thesis is to detect parasite DNA in the serum and cerebrospinal fluid of infected ducks, and also in the serum of infected mice. By using PCR with primers designed for a tandem repeated sequence, we were able to detect 1 femtograms of parasite DNA isolated from sera of infected ducks. We were able to amplify parasite DNA only from 16 samples of sera and cerebrospinal fluid of the infected ducks, but we were not able to do so with the serum of mice. Sera of infected mice were also tested by ELISA and Western blot. The homogenates of T. regenti (TRhc), T. szidati (TShc) and S. mansoni (SMhc) cercariae were selected as an antigen. The results showed progressive increase in the level of IgM antibody from 10 days after 1st infection and also increase of the level of IgG from the 2nd infection. 10 days after the 4th infection, the level of IgM and IgG gradually declined, but the level of antibodies 100 days after the 4th infection was still higher if compared to uninfected mice. Results from Western blot analysis...
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Molecular diagnostics of bird schistosomes during the infection of natural and accidental hosts
Šteiger, Vladimír ; Kašný, Martin (advisor) ; Dvořák, Jan (referee)
Bird schistosomes of the genus Trichobilharzia are known as causative agents of hyper-immune skin reaction called cercarial dermatitis (swimmer's itch). They use pulmonary water snails from family Lymnaeidae as the intermediate host and mostly anatid birds as the definitive host. The first larva, miracidium, actively moves in water environment, penetrates the snail and develops to the mother sporocyst. Then the daughter sporocysts are formed and migrate to the hepatopancreas of the snail where the high number of cercariae is assexually produced. Cercariae leave the intermediate host, actively move in a water and penetrate the skin of definitive host. Within a host body they mature and lay eggs. Cercariae can penetrate also the mammalian skin, including human, where they are immediately eliminated by the immune system of the host, which is followed by inflammatory reaction. Until now, for humans, there is no effective method enabling to differ cercarial dermatitis from other hyper-immune skin reactions and for birds the reliable diagnostic method of trichobilharziasis is missing. The main aim of this thesis was to use the molecular methods for diagnostic of bird schistosomes infection in natural (ducks) and accidental hosts (mice, human). For optimization, the conventional PCR was used for detection...
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Aporocotyle simplex fluke from the flounder Hippoglossoides platessoides in Svalbard.
HÁJKOVÁ, Šárka
In my bachelor thesis I studied trematodes from plaice Hippoglossoides platessoides obtained during expeditions to Svalbard in 2014 and 2018. My goal was morphological and molecular processing of the obtained material, which allowed me to confirm the identification of the flukes as Aporocotyle simplex Odhner, 1900. Aporocotyle simplex is a common parasite of plaice, ocurring mainly in Hippoglossoides platessoides, but also in Limanda limanda and Pleuronectes platessa. The species A. simplex is geographically distributed in the Gulf of St. Lawrence in Canada, off the west coast of Sweden, in the Barents Sea and Bering Sea, off the coast of Kamchatka and Greenland. My results showed that the species A. simplex is widespread in Svalbard. In the practical part, I dealt with methods that helped me to identify the studied specimens. I observed the trematode tegumental surface with aid of a scanning electrom microscope (JEOL JSM-7401F). The trematode internal organs were stained with Mayer-Schuberg carmine, which allowed me to observe and draw the mounted specimens using a light microscope (Olympus BX51) equipped with a drawing attachment. Furthermore, for molecular analysis, the DNA was isolated using the commercial Exgene Tissue SV mini kit (GeneAll). The isolated DNA (the gene for the large ribosomal subunit, 28S rRNA) was amplified by PCR, the obtained PCR products were sequenced on an automated sequencer (ABI Prism 3130xl or 3730xl, by SEQme). The newly acquired sequences (three in total) were aligned in the program Geneious 8.0.5with sequences available from GenBank (64 sequences). Phylogenetic analysis of the species relationships within the family Aporocotylidae was performed using the Maximum Likelihood method with TPM2u + F + G4 used as the best model. Aporocotyle simplex falls into a separate branch together with A. michaudi, from which however, it differs substantially by its morphology (distribution of tegumental spines on body and their number in the clusters, number of testes, ratio of esophagus length to body length) and geographical distribution (A. michaudi occurs in the South Atlantic Ocean).
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Detection of Ralstonia solanacearum bacteria pathogenic for tomato, pepper and potato by loop-mediated isothermal amplification
STRNAD, Adam
For rapid detection of quarantine bacteria Ralstonia solancearum pathogenic for tomato, potato and papper, the LAMP method was used. Based on the analysis of the fliC gene sequence, a set of LAMP primers was designed to be specific for Ralstonia solanacearum. Subsequently, the method was optimized to detect only Ralstonia solanacearum and to distinguish it from the other phytopathogenic bacteria which infect potato, tomato and pepper. LAMP can detect bacteria from very small amounts of DNA in less than 30 minutes in real time. The method is capable of amplifing DNA at a concentration of 0.00001 ng/?l. However, a higher DNA concentration is more suitable for more specific detection.
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Molecular diagnostics of bird schistosomes during the infection of natural and accidental hosts
Šteiger, Vladimír ; Kašný, Martin (advisor) ; Dvořák, Jan (referee)
Bird schistosomes of the genus Trichobilharzia are known as causative agents of hyper-immune skin reaction called cercarial dermatitis (swimmer's itch). They use pulmonary water snails from family Lymnaeidae as the intermediate host and mostly anatid birds as the definitive host. The first larva, miracidium, actively moves in water environment, penetrates the snail and develops to the mother sporocyst. Then the daughter sporocysts are formed and migrate to the hepatopancreas of the snail where the high number of cercariae is assexually produced. Cercariae leave the intermediate host, actively move in a water and penetrate the skin of definitive host. Within a host body they mature and lay eggs. Cercariae can penetrate also the mammalian skin, including human, where they are immediately eliminated by the immune system of the host, which is followed by inflammatory reaction. Until now, for humans, there is no effective method enabling to differ cercarial dermatitis from other hyper-immune skin reactions and for birds the reliable diagnostic method of trichobilharziasis is missing. The main aim of this thesis was to use the molecular methods for diagnostic of bird schistosomes infection in natural (ducks) and accidental hosts (mice, human). For optimization, the conventional PCR was used for detection...
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Molecular and serologic diagnosis of infections caused by Trichobilharzia
Vaščiková, Michaela ; Horák, Petr (advisor) ; Ditrich, Oleg (referee)
Cercariae of the genus Trichobilharzia can penetrate not only the skin of definitive hosts (ducks), but they are also able to penetrate the skin of accidental hosts (mammals). As a result of the penetration, the inflammatory response known as cercarial dermatitis appears. The goal of our thesis is to detect parasite DNA in the serum and cerebrospinal fluid of infected ducks, and also in the serum of infected mice. By using PCR with primers designed for a tandem repeated sequence, we were able to detect 1 femtograms of parasite DNA isolated from sera of infected ducks. We were able to amplify parasite DNA only from 16 samples of sera and cerebrospinal fluid of the infected ducks, but we were not able to do so with the serum of mice. Sera of infected mice were also tested by ELISA and Western blot. The homogenates of T. regenti (TRhc), T. szidati (TShc) and S. mansoni (SMhc) cercariae were selected as an antigen. The results showed progressive increase in the level of IgM antibody from 10 days after 1st infection and also increase of the level of IgG from the 2nd infection. 10 days after the 4th infection, the level of IgM and IgG gradually declined, but the level of antibodies 100 days after the 4th infection was still higher if compared to uninfected mice. Results from Western blot analysis...
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