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Methods for diagnosing SARS-CoV-2 virus
HELMICHOVÁ, Eva
This bachelor thesis deals with the study of SARS-CoV-2 virus diagnostics. It is an RNA coronavirus that causes COVID-19. It was discovered in late 2019 in China and then the disease spread around the world. This disease is manifested by severe cough, loss of taste and smell. In worse cases, it manifests as pneumonia.Diagnosis of SARS-CoV-2 virus is performed using PCR assays, antigenic assays, and antibody assays. Examination with antigenic tests is intended for rapid screening for the presence of the virus. The test for the detection of antibodies is suitable for verifying the disease, or we find out the effectiveness of vaccination and resistance to it. These samples are obtained by swabbing the nasopharynx, mouth from saliva or blood. Sampling should be correct and thorough for SARS-CoV-2 virus analysis.The PCR method is considered to be the most reliable method for diagnosing SARS-CoV-2 virus. The method allows us to detect the virus on the basis of known sequences and, using suitable primers, the individual variants of the viruses are distinguished. Morphology, diagnostics of SARS-CoV-2 virus and PCR methodology with precise primer design for SARS-CoV-2 detection are presented in this work. Using software a comparison of SARS-CoV-2 genomes in a given mutated region and selected sites in which they differ from each other is performed.
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β-tubulin paralogs in Aspergillus: taxonomical importance and molecular tools for distinguishing
Hubka, Vít ; Kolařík, Miroslav (advisor) ; Bunček, Martin (referee)
A beta-tubulin gene (benA) is widely used in taxonomy and identification of Aspergillus spp. and other Fungi.Across Aspergillus spp. There is either one (benA) or two beta-tubulin paralogs (benA and tubC). The risk ofcontemporary use of sequences of paralogous genes with non-homologous function in the same phylogeneticanalysis is well known. It is evident that it had happened repeatedly in Aspergillus section Nigri. It is alarmingthat conventional primers for amplification of partial benA sequence can specifically amplify tubC paralog insome species. In this work, both paralogs were characterised in a set of species. The beta-tubulin primers in usewere revised and new, more benA specific primers were designed. Applicability of some markers such as basecomposition, codon usage and length of introns for distinguishing -tubulin paralogs benA and tubC is tested. Alarge study on molecular diversity of 349 isolates of Aspergillus (PCR-fingerprint, sequence data - ITS, benA,rpb2, caM) originating from Czech culture collections and from clinical material is also included. 82 specieswere identified, togetherwith nine tentative new taxa belonging to sections with high economic impact - Nigri,Fumigati or Aspergillus (Eurotium spp.). Five species from Section Aspergillus could be synonymised withexisting taxa. A study...
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Optimization of PCR detection of biosynthetic alkaloid pathway genes in opium poppy
BRÁZDOVÁ, Sára
The thesis deals with the creation of the biosynthetic pathways of selected alkaloids of opium poppy. Then is following the design primers for selected genes and optimization of PCR reaction of these genes for amplification and sequencing of the longest fragments. The PCR reaction was optimized for the 7OMT, TNMT and CODM genes. These genes are involved in biosynthetic pathways of morphine, codeine, papaverine, noscapine and sanguinarine. Each gene was split in half. The primers were designed separately for each half of the gene. Altogether were designed 13 pairs primers. 5 pairs primers were optimized by gradient PCR and gel electrophoresis. These primers were used for sequencing analysis.
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Detekce a monitoring potenciálně toxických sinicových lipopeptidů
BÁRTOVÁ, Marie
The aim of this study was to design and optimize new PCR primers for detection of potential cyanobacterial producers of cytotoxic lipopeptides puwainaphycins and minutissamides in environmental samples. Samples from two distinct localities were tested, as suggested based on preliminary data. The first set of samples consisted of cyanobacterial soil biofilms from sheep pastures affected by Alveld illness in Norway. The other one contained samples of planktic cyanobacaterial blooms from Protected Landscape Area Třeboň and its vicinity. Three different approaches were used for evaluation of the presence of cyanobacterial lipopeptide producers: microscopy, PCR with the designed primeres, and liquid chromatography-mass spectrometry analysis. Results of this study confirmed the specificity of the newly designed PCR primers. The presence of producers of puwainaphycins/minutissamides was proven at both tested localities.
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β-tubulin paralogs in Aspergillus: taxonomical importance and molecular tools for distinguishing
Hubka, Vít ; Kolařík, Miroslav (advisor) ; Bunček, Martin (referee)
A beta-tubulin gene (benA) is widely used in taxonomy and identification of Aspergillus spp. and other Fungi.Across Aspergillus spp. There is either one (benA) or two beta-tubulin paralogs (benA and tubC). The risk ofcontemporary use of sequences of paralogous genes with non-homologous function in the same phylogeneticanalysis is well known. It is evident that it had happened repeatedly in Aspergillus section Nigri. It is alarmingthat conventional primers for amplification of partial benA sequence can specifically amplify tubC paralog insome species. In this work, both paralogs were characterised in a set of species. The beta-tubulin primers in usewere revised and new, more benA specific primers were designed. Applicability of some markers such as basecomposition, codon usage and length of introns for distinguishing -tubulin paralogs benA and tubC is tested. Alarge study on molecular diversity of 349 isolates of Aspergillus (PCR-fingerprint, sequence data - ITS, benA,rpb2, caM) originating from Czech culture collections and from clinical material is also included. 82 specieswere identified, togetherwith nine tentative new taxa belonging to sections with high economic impact - Nigri,Fumigati or Aspergillus (Eurotium spp.). Five species from Section Aspergillus could be synonymised withexisting taxa. A study...
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Design and testing of multiplex-PCR primers for detection of bacterial spot of tomato
STEHLÍKOVÁ, Dagmar
The subject of this work is to develop multiplex-PCR assay for specific detection of plant pathogenic bacteria of Xanthomonas genus causing bacterial spot of tomato. PCR primers for detection of groups A (X. euvesicatoria), B (X. vesicatoria), C (X. perforans) and D (X. gardneri) were developed based on the DNA sequences obtained by sequencing and from the GenBank database (NCBI). Four primer pairs - Xe_shotgun_104, Xe_shotgun_1819, Xv_atpD_403, Xp_efP_202 were designed and subsequently thoroughly tested and optimized for parallel detection of these bacteria. Specificity of the primers was tested on a large complex of bacterial strains pathogenic to tomato and related crops. Following the protocol described above X. vesicatoria, X. euvesicatoria, X. perforans and X. gardneri can be quickly and reliably identified in a single multiplex-PCR assay.
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