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Analysis of metabolome effects of hop (H. lupulus) transcription factors in heterologous of Petunia hybrida
MORAVCOVÁ, Vendula
Plant transformation is now a key research in plant biology, and also is a unique practical tool in the improvement of varieties of cultivated plants. The aim of this work was to obtain transgenic plants Petunia x hybrida containing transgene nptII (kanamycin resistance gene) using the indirect transformation by Agrobacterium tumefaciens. In terms of in-vivo was grown from seeds experimental material in the form of plant Petunia x hybrida cv. Andrea. For the experiments three different constructs HLWD40 3278-80 and HLbHLH 3577 and HLbHLH 3677 GFP were used. The leaf explants from Petunia plants were prepared. Explants were transferred using tweezers into the crucible containing liquid ? MS and prepared bacteria were added. Thus prepared explants remained in bacteria the next day, when they were transferred to the regenerating solid ground. The explants formed callus gradually (clusters of cells) which regenerate a new plant. Transformation should satisfy the condition that the transformed plants should be fertile. For this reason, an attempt was made crossing the transformed plants were crossed Pap1 1527/2 and Pap1 1572/4 with plants crossed HLWD 40 3278-80. The grown plants were analysed by a PCR reaction. The explants began during the first 2-4 weeks after transfromation to form callus and during the next week to remove the first regenerants. At one exulant fell averaged around 7 regenerants and the explants regenerated by construct HLbHLH 3577 regenerate better than regenerants transformed with construct HLWD 40 3278-80. During the transformation we obtained regenerants. With "tissue" PCR, it was found that 15 plants carried the gene for kanamycin resistance. Transformation efficiency was 3.75%.
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The diagnostics of the hepatitis C virus with usage of methods of the molecular biology
JAKUBCOVÁ, Markéta
Hepatitis C is a virus caused liver irritation, which leads to hepatocelular carcinoma in more than a quarter of cases. Six different genotypes with at least fifty different subtypes are known (3). Hepatitis C virus rapidly undergoes mutation, which causes every infected person to have more of slightly different variants of the virus in their body. Consequently, the immune system often loses control over the virus, which can lead to the observed chronicity. Qualitative confirmation is important for determination of the presence of the virus. Quantitative confirmation, on the other hand, is crucial for commencing and monitoring of the treatment. (20) The most often examined species is blood serum, or plasma. HCV can be diagnosed directly, or indirectly. Confirmation of the presence of anti-HCV antibodies (indirectly) by Elisa method is routine and very basic screening in most laboratories. Detectable anti-HCV antibodies occur three weeks to six months after the primoinfection (every person?s immune system reacts differently). This indirect test is usually negative in early phases of infection. (21) For direct confirmation of the virus, solely PCR ? polymerase chain reaction ? methods are used. In the first phase, it is necessary to isolate RNA of the virus from the biological material. This can be done either by a special isolation instrument, or manually, using special isolating kits. Use of different methods may result in different yield and purity of RNA. In my work, I used only hand column isolation. After obtaining the RNA of the virus, qualitative confirmation follows. In my case, an ?In house? method was used ?the RT PCR (reverse transcription PCR) followed by the PCR amplification of specific segment of viral RNA, which are visualized by gel electrophoresis in presence of ethidium bromide, which works as an intercalation species. Double step PCR is used. In the first step, reverse transcription progresses for 30 minutes at 50°C. In the presence of RNase inhibitor and reverse transcriptase enzyme RNA is overwritten to cDNA, which is subsequently multiplicated using specific primers. This process is a part of one PCR reaction, which takes place in the Thermocycler in the presence of specific primers, water, buffer, nucleotides, and specific enzyme. For the second round of Nested PCR two different sequences of primers are used. PCR is used for enhancing the sensitivity and specifity of the method. Products of the PCR are then visualized on the agarose gel with intercalation agent (most commonly ethidium bromide).(3) Quantitative determination of viremia level is carried out with Real Time PCR. Detection proceeds in real time using fluorescence dyes. Dyes are usually bonded onto oligonucleotide probe, which specifically bonds to a PCR amplificate. Measuring the intensity of fluorescence enables confirmation and quantification of the products. The system is also able to identify potencial PCR inhibition with internal control. After addition of standards of known concentration, we can work out a calibration curve and use it to quantify measured samples (19). In this work, two isolation kits are compared: a) High Pure Viral RNA Kit (Roche) ? kit used for isolation viral RNA from biological material (serum, plasma). In the first phase, biological material is decomposed, RNA is collected on the column with membrane, washed multiple times and eluted to water for use in Real Time PCR (8) b) QIAamp Viral RNA mini Kit (Qiagen) ? kit used for isolation of viral RNA from blood plasma, or serum. Firstly, biological material is decomposed, RNA is collected on the column with membrane, washed multiple times and eluted to water for use in Real Time PCR (13).The samples isolated with the QIAamp Viral RNA Kit (Qiagen) have shown higher values of measured viral RNA after quantification.
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Diagnostics of Clostridium difficile as nosocomial infections in Nemocnice ČB a.s. using methods of molecular biology.
ŠTĚRBOVÁ, Denisa
Nosocomial infections caused by Clostridium difficile represent a substantial part of hospital-acquired infections in Czech hospitals. Intoxication by toxins produced by Clostridium difficile leads to serious damage to gastrointestinal tract and life of the patient may be in danger. The progress of intoxication could be quick, this is why reliable and time-efficient diagnostic methods are of great importance for efficient treatment of the patients. Bacterial toxins are not produced during the whole life cycle of Clostridium difficile. This is why it is better to detect bacterial DNA which is always present in the bacterial cells, not the toxins. In Nemocnice ČB a.s. (České Budějovice municipal hospital) I compared methods based on toxins detection (?hyplex? ClosTox? and ?hyplex? ClosTox 027? by BAG Health Care) with a method based on DNA detection (real-time PCR ?Xpert C. Difficile? by Cepheid). I found out the real-time PCR method is much quicker. It takes one hour to prepare the samples and to obtain analytical result for this method. Both tests based on toxin detection are much more time consuming. It takes up to 5 hours to complete them. I conclude the real-time PCR is much quicker analytical method and it allows Clostridium difficile detection during all life phases of the bacteria.
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Endoparasitosis of feathered game
BARVÍŘ, Pavel
A total of 180 samples, 119 from pheasant (Phasianus colchicus) and 61mallard (Anas platyrhynchos) were collected during two consecutive years (from 2011 to 2012). All samples were examined for the presence endoparasite using the flotation method according Sheather. In samples from pheasants were detected these types of parasites: Capillaria spp., Eimeria spp., Heterakis spp., and Trichostrongylus spp. In addition, Syngamus spp. and Cryptosporidium spp. were detected in the duck samples. Microscopical examination of aniline-carbol-methyl violet stained fecal smears revealed to Cryptosporidium spp. 12 positive samples originating from Anas platyrhynchos. DNA was extracted from Cryptosporidium positive samples and all microscopically negative samples. Nested PCR was performed to amplify the partial SSU rRNA gene of Cryptosporidium. The sequence analyses of PCR-positive specimens identified 10 samples as Cryptosporidium avian genotype III and 4 samples as C. muris. No clinical signs were detected in any Cryptosporidium positive animals. This is the first report of Cryptosporidium avian genotype III in the Czech Republic and also in Anas platyrhynchos.
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Congenital disorders by pigs
MUSILOVÁ, Dagmar
This diploma thesis follows the bachelor thesis and it?s task is to processing analysis of the occurrence of PSE meat in a population of the Czech white breed as a pigs stress syndrome impact. General problem of congenital diseases is processed in literature review. Further, this work deals with pigs stress syndrome and stress, which causes the meat defects as an external factor. Molecular - genetic methods, which are used in the practical part, are described as well. The research focused on genotyping of loci selected panel of samples, to determine the frequency of alleles and genotypes of selected locus and the statistical relationship between genotype and expression of PSE meat. Results were statistically processed and evaluated at the end.
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Molecular and biochemical characteristics of genetically modified barley plants
KOCKOVÁ, Lucie
Modern agriculture often employs broad-spectrum herbicides combined with herbicide-resistant crops. Usually, this is achieved by altering the crop genom (genetic modification) by inserting of a specific gene coding for resistance to specific herbicide or group of herbicides. Apllying such herbicide thus results in minor crop damage. The resistence of crops against broad-spectrum herbicides depends on the genes, which were inserted into the genom. The gene bar is often used as a resistence -providing element. It mediates resistance against a broad spectrum of herbicides, such as glufosinate and Bialahops. For the selection of transformants and preliminary assessment of the target transgen expression level a suitable marker gene is simultaneously inserted. For this purpose, gene for bacterial ??glucuronidase (GUS) is often used. It is considered to be one of the most frequently used reporter systems for the assessment of transgen expression and also allows to analyze the expression on the tissue, cell and whole cell organelles levels. In this diploma thesis the PCR method and histochemical detection of the enzyme glucuronidase presence were used to detect and evaluate transgenic plants of cv. Golden Promise spring barley, modified by genes bar and gus. The presence of gus gene was determined in different parts of plants.
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Methods of DNA extraction from the fresh papaya fruit and from the candied
Ovesná, Jaroslava ; Hodek, Jan
Aim the work is to provide a working procedure for extraction of amplifiable DNA form papaya fruits (Carica papaya), which are a poor source of DNA. Methods are optimised for DNA extraction from papaya fruits and stones and from candied papaya- these both were predicted as suitable commodities for an eventually screening of non-approved GM papaya in the market of Czech Republic. The methodology was prepared on basis of demand of reference laboratories of public service, which are involved in GMO analyses and also as a flow-work of Methodics for Detection of GM papaya 55-1 and 63-1 (Hodek, Ovesná, Pavlátová 2008) and scientific publication Detection of transgenic papaya lines: extraction protocol optimisation and verification of DNA quality by PCR assay (Ovesná, Hodek 2009).
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