>Magister Scientiae - MSc / There are limited reports on virus population in haloalkaline environments; therefore the aim of this study was to investigate the genetic diversity and biology of bacteriophage communities in these environments. Bacteria were isolated to be used as phage hosts. One bacterium from Lake Magadi and four bacteria from Lake Shala were successfully isolated from sediment samples. A further two Lake Shala bacterial hosts from the IMBM culture collection were also used to isolate bacteriophages. Bacterial isolates were identified to be most closely related to Bacillius halodurans, Halomonas axialensis, Virgibacillus salarius, Bacillus licheniformis, Halomonas venusta, Bacillus pseudofirmus and Paracoccus aminovorans. Bacteriophages were screened using all bacteria against sediment samples from both Lake Shala and Lake Magadi. One phage was identified from Lake Magadi sediments (MGBH1) and two phages from Lake Shala sediments (SHBH1 and SHPA). TEM analysis showed that these phages belong to three different dsDNA phage families; Siphoviridae (MGBH1), Myoviridae (SHBH1) and Podoviridae (SHPA). All phages showed different genome sizes on agarose gel. Due to the small genome size, phage SHPA was chosen for further investigation. Partial, genome sequence analysis showed homology to both bacterial and phage proteins. A further investigation of phage diversity in this environment is essential using metagenomic approaches to understand these unique communities.

The aim of work was an optimization of separation deoxyribonucleic acid (DNA) with the use of nucleic acid reversible adsorption to the surface of magnetic particles coated by functional groups. Six carriers were verificated for DNA isolation: P (HEMA-co-GMA) ox, F-kol B 30 ox, F-kol 77 ox, F-kol B100 ox, F-kol 135 ox, coated with carboxyl groups and Perovskit 439 (coated by silicone). Bacterial DNA was isolated by phenol extraction procedure, first. DNA was reversibly bond to magnetis carrier in the presence of high concentration of NaCl ( 5 M) and poly (ethylene glycol) (PEG 6000). The final PEG and NaCl concentrations of 16.0 % (w/v) and 2.0 M, respectively, were used.DNA was eluted into TE buffer. The quality of extracted DNA was checked by PCR amplification. It was found out that although different quantities of DNA were isolated, the quality of isolated DNA was always compatible with PCR. Nanoparticles Perovskit 439 had the best separative characteristics in comparison to the other magnetic carriers because highest amounts of DNA was isolated. However, next optimisation of DNA separation procedure is required for the use of studied microspheres in real samples.

Lactic acid bacteria are natural habitants of human gastrointectinal tract. Among the most important are bacteria of genus Lactobacillus and genus Bifidobacterium that contain a lot of probiotic species. Probiotic species are used as food supplements. This work was focused on DNA separation from crude cell lysates of 4 food supplements using magnetic carrier P(HEMA-co-GMA) covered by carboxyl groups. DNA was reversible adsorbed to the carriers in the presence of PEG 6000 (16%) and NaCl (2 M) (final concentrations) and eluted into TE buffer. Lysis of cells from food supplements was performed by lysozyme, SDS and proteinase K. The amount of lysozyme was optimalized. Concentration of separated DNA was measured by spectrophotometric method. The amount of isolated DNA was suitable for PCR. Isolated DNA was used for PCR with universal primers, PCR specific for genus Lactobacillus and genus Bifidobacterium and for 9 different species-specific PCRs: Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei/paracasei, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium infantis. Amplicons were detected by agarose gel electrophoresis (1,8%). It was shown that DNA amplification methods are quick and precise for identification of studied species. The results of bacteria identification were compared with data provided by the manufacturer. In all food supplements, bacteria of genus Lactobacillus and Bifidobacterium were detected. However, only some species provided by manufacturer were identified by PCR in each tablet.

Lactobacilli are dominant bacteria of the vaginal flora. Lactobacillus-containing probiotics products are used for the treatement and profylaxis of bacterial urogenital infections. This work is focused on DNA identification and species identification of probiotic bacteria in 5 different vaginal tablets using molecular-genetic methods. Total DNA isolated from complex matrix of vaginal tablets was used for amplification in polymerase chain reaction. DNA was isolated from crude cell lysates by magnetic particles P(HEMA-co-GMA) and by method of phenol extraction. Identification of species of probiotic bacteria was verified using genus-specific and species-specific PCRs. Results of bacterial identification obtained by PCR were compared with declared specification given by producers. Bacteria of genus Lactobacillus were proved in all tablets whereus species identification was in accordance with the stated composition in 1 tablet only.

In the food industry are requested speedy and accurate methods for identification of bacteria in microbiological testing of products. Molecular diagnostic methods are based on isolation of DNA from bacterial cells which is amplified in polymerase chain reaction (PCR). The result is fragment DNA about specific size, characteristic for genus or species of bacteria. The aim of the work was isolation of PCR-ready DNA. DNA has been isolated from 8 strains of genus Clostridium. Procedure of cell lysis was optimized in order to find the optimal concentration of EDTA and proteinase K in lysing buffer. DNA was isolated by phenol extraction and using magnetic microspheres. Concentrations 10 mM of EDTA and 10 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by phenol extraction. Concentrations 10 mM of EDTA and 15 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by magnetic microspheres. Isolated DNA was checked by gel electrophoresis, quantificated by spectrophotometry and tested in PCR. Individuals species were distinguished in denaturing gradient gel electrophoresis (DGGE).

Biological evidence may contain any number of cells in any proportion. Extreme low-template DNA samples are often very difficult to interpret due to complex signal or peaks which may be indistinguishable from baseline noise. Current solutions focus on increasing the amount of amplicon detected by adjusting PCR cycle number or capillary electrophoresis injection parameters. Consensus profiling is an additional option. However, the aforementioned solutions are often not helpful for extreme low-template samples due to the high occurrence of allelic drop-out. Additionally, PCR is a destructive technique that causes one amplification to completely exhaust this type of sample, making further typing and analysis impossible. Therefore, a technique that allows for the re-generation of a DNA template in order to amplify it multiple times would be an extremely useful tool. This study outlines the development of a method that allows for the recursive amplification of a DNA sample. Amplification was performed using biotinylated primers for an STR locus and the resulting product was cleaned using streptavidin-coated magnetic beads to sequester the amplicons. Subsequent centrifugal filtration was used to remove the remaining PCR components, thus isolating the original genomic DNA. Re-amplification was then successfully performed at a different STR locus. Though successful, multiple run-throughs of the method indicated retention of signal from the original amplification as well as significant genomic DNA loss during the process. This study outlines experiments seeking to characterize the cause(s) of these imperfections in order to effectively direct method optimization. A computer generated dynamic model was also created and used to simulate the recursive amplification process to assist in development. When optimized, it is expected that recursive amplification can significantly reduce the difficulties associated with low-template DNA analysis and eradicate the concept of an ‘exhaustive’ DNA sample.

The theoretical part summarizes information on the isolation and purification of plasmid DNA and nucleic acids as. Plasmid DNA is often used in gene engineering as a vector for the transfer of a particular gene. Its insulation and transportation in sufficient quality is crucial for other processes associated with it. Isolation and survival of pDNA using magnetic carriers of different concentrations of PEG 8000 in combination with 1M NaCl was investigated in experimental parts. Furthermore, the isolation of pDNA using commercial kits was examined.

<p>Bacillus anthracis is one of the most feared agents of biological warfare and causes the</p><p>deadly disease called anthrax. SVA (statens veterinärmedicinska anstalt) is working on a</p><p>project together with SLV (statens livsmedelsverk) where the target is to find rapid and</p><p>effective detection methods for Bacillus anthracis in animal feed and food samples. Bacillus</p><p>subtilis, which is harmless, was used in this study as a model organism to Bacillus anthracis.</p><p>A known concentration of vegetative Bacillus subtilis was spiked in animal feed and food</p><p>samples. The genomic work flow was based on automated DNA isolation and real time PCR.</p><p>The aim of the study was to screen for inhibitory components in the animal feed and food</p><p>samples using two different DNA isolation robots; Magnatrix 8000 and Biorobot EZ1. The</p><p>results showed that DNA of high quality was extracted from the samples with both robots.</p><p>However, the CT-value generated by the real time PCR showed considerable variation</p><p>depending on the sample matrix. Some samples, for instance egg and liver, were problematic</p><p>and gave low concentrations and high CT-values probably due to inhibitory components in the</p><p>samples. Further studies will be needed to solve these problems and optimize the methods that</p><p>were used in this study.</p>

Bacillus anthracis is one of the most feared agents of biological warfare and causes the deadly disease called anthrax. SVA (statens veterinärmedicinska anstalt) is working on a project together with SLV (statens livsmedelsverk) where the target is to find rapid and effective detection methods for Bacillus anthracis in animal feed and food samples. Bacillus subtilis, which is harmless, was used in this study as a model organism to Bacillus anthracis. A known concentration of vegetative Bacillus subtilis was spiked in animal feed and food samples. The genomic work flow was based on automated DNA isolation and real time PCR. The aim of the study was to screen for inhibitory components in the animal feed and food samples using two different DNA isolation robots; Magnatrix 8000 and Biorobot EZ1. The results showed that DNA of high quality was extracted from the samples with both robots. However, the CT-value generated by the real time PCR showed considerable variation depending on the sample matrix. Some samples, for instance egg and liver, were problematic and gave low concentrations and high CT-values probably due to inhibitory components in the samples. Further studies will be needed to solve these problems and optimize the methods that were used in this study.

The hereditary cataract is one of the most common eye disease in dogs. The expansion of this disease in the Staffordshire bullterrier breed has been so massive that in the Czech Republic was introduced the rule of mandatory testing of at least one of a breeding pair. This is a degenerative disease of the lens causing total blindness of the affected animal within three years. Since some time ago there are no more dogs affected by the disease in the Czech Republic, there are however still hidden carriers which need to be discovered to the complete extinction of the disease in the genome. The goal of this study was to test simple ways of collecting biological samples, try them in practice and to verify whether they are suitable for the DNA isolation and also to test an alternative method of molecular detection of this disease. In total there have been 23 buccal swabs collected from male and female Staffordshire Bullterrier examples. The detection of the hidden carriers of the hereditary cataract was carried out by PCR analysis with specific primers. The obtained amplicons were detected by both gel and chip electrophoresis and by using fragment analysis. This detection of the carriers was based on the presence of two amplicons (heterozygotes). I came to conclusion that to detect hidden carriers it is neccessary to use the fragment analysis because of the difference of only one base in the reference section of DNA. Neither gel nor chip electrophoresis does provide sufficiently high resolution and it is not possible to detect two fragments that differ only by one bp. As the most appropriate sampling method I have chosen the buccal smear by cytological brush followed by isolating the DNA by Chelex with purification of the sample subsequently.