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Efekt polynenasycených mastných kyselin n-3 ve výživě potkana na expresi vybraného genu
Zamazalová, Nikola
The aim of my thesis on topic The effect of n-3 polyunsaturated fatty acids in the diet of rats on expression of selected genes was to investigate the effect of eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) on the expression of genes which encode GPR120, AdipoR1 and AdipoR2 receptors in relation to suppress low-grade chronic inflammation in organism to reduce the risk of atherosclerosis by dietary intervention in rats. Rats were fed by a mixture MYPO with 6 % safflower oil (diet S), 6 % fish oil (diet F) or 6 % of oil from algae Schizochytrium (diet A). Gene expression was measured by quantitative real-time PCR method and the results were evaluated by using the software qbase + (Biogazelle NV). Relative expression of GPR120 gene was in F diet 88 % (P > 0,05), in A diet 93 % (P > 0,05) in comparison with control group (100 %). Relative expression of ADIPOR1 gene was in F diets and A diet 82 % (P < 0,05) in comparison with the control group. For ADIPOR2 gene relative expression was 71 % (P < 0,05) in diet F and 68 % (P < 0,05) in diet A. The results were contrary to our hypothesis. However, they exactly match the results of other studies in the available literature. It would be appropriate to carry out further studies on this issue.
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Application of the method PCR-HRM analysis to identify bacteria in foods and food supplements
Šurková, Alice ; Illková, Kateřina (referee) ; Trachtová, Štěpánka (advisor)
Theoretical part of the thesis was focused on foods and food supplements containing microorganisms, especially bacteria. Furthermore, the thesis deals with methods for identification of the bacteria, primarily polymerase chain reaction (PCR). The thesis also includes real-time PCR and is specially focused on high resolution melting analysis (HRM). During the experimental part, the DNA sample was isolated from a chosen probiotics product using magnetic microparticles. The concentration of the DNA sample was determinate and DNA was subjected to PCR with subsequent detection PCR products by agarose gel electrophoresis. To the results specify HRM analysis was then performed.
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Identification DNA of Plant and Animal Species in Food by Polymerase Chain Reaction
Šmíd, Jiří ; Hanák,, Petr (referee) ; Timko,, Jozef (referee) ; Kuchta, Tomáš (advisor)
We were developing detection methods for three food allergens of plant origin. We used real-time PCR for soy detection in food oriented on gene lec, that is coding lektine specific for soy. On this target sequence were oriented PCR system with primers Le2F and Le2R and TaqMan probe Le2P. Detection limit (2,75 pg), practical detection limit (0,02 %), inclusivity and exclusivity were determined. Whole system were quantified. Real-time PCR for pistachio detection were based on primers and probe for gene COR. Detection limit (3,5 pg), practical detection limit (0,002 %), inclusivity and exclusivity were determined. For almond detection we were not succeed system, that fulfil all qualitative parametres.
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Foodborne Staphylococcus Aureus: Identification and Enterotoxin Production in Milk and Cheese.
Hrušková, Vendula ; Španová, Alena (referee) ; Kmet,, Vladimír (referee) ; Kaclíková, Eva (advisor)
Onemocnění z potravin (alimentární onemocnění) vyvolaná bakteriemi jsou stále aktuálním tématem v celosvětovém měřítku. Abychom zajistili výrobu zdravotně nezávadných potravin, je potřeba nových poznatků o virulenci patogenů, které by doplnily již známé skutečnosti o jejich růstu a přeživání v potravinách. Také potřebujeme vyvíjet rychlé a citlivé metody na detekci těchto patogenů. Dizertační práce popisuje metodu na detekci S. aureus v potravinách, která je založená na PCR v reálném čase ve spojení s namnožením v selektivním médium. Dále pojednává o vlivu environmentálních faktorů na růst S. aureus a tvorbu enterotoxinů v mléce a sýrech. Vyvinuli jsme rychlou a citlivou metodu na detekci S. aureus v potravinách s použitím selektivního namnožení a PCR v reálném čase. Nově vyvinutá metoda umožnila detekci S. aureus na druhý den od přijetí vzorku. Tato metoda může být použita jako rychlejší, citlivějsí a vysoce specifická alternativní metoda ke konvenční mikrobiologické metodě. Zkoumali jsme vliv tří různých teplot, 8°C, 12°C a 20°C na růst S. aureus a tvorbu enterotoxinu D v pasterizovaném mléce a na růst, expresi genu sed a tvorbu enterotoxinu D v tekutém médiu s extraktem z mozku a srdce (BHI). Experimenty byly prováděny v malých skleněných fermentorech po 6 dní. Genová exprese byla sledována pomocí qRT-PCR a tvorba enterotoxinu D byla měřena pomocí imunologické metody ELISA. Růstová křivka v BHI měla stejný průběh při 20°C a 12°C, ale v při 12°C začal růst se spožděním. Při 8°C nebyl pozorován žádný růst. Růst S. aureus v mléce byl ve srovnání s BHI menší. sed mRNA byla detekována při 20°C po 4 hodinách a při 12°C po 7 hodinách a produkce enterotoxinu se objevila v exponenciální fázi růstu. V mléce se produkce SED při 20°C a při 12°C objevila dříve, ale celkové množství vyprodukovaného SED bylo nižší než v BHI. Při 8°C nebyla pozorována žádná produkce SED stejně jako v BHI. Dále byl zkoumán společný vliv nízké teploty 12°C a přítomnosti kompetitivní doprovodné mikroflóry pocházející ze surového mléka na růst S. aureus a produkci enterotoxinu v pasterizovaném mléce. Byl pozorován inhibiční účinek na růst a produkci enterotoxinů a vliv kompetice byl výraznější než vliv nízké teploty. Produkce enterotoxinu byla nízká a odpovídala růstu. Snížením množství doprovodné mikroflóry a zvýšením inokula došlo pouze k nepatrnému zvýšení produkce enterotoxinu. V další fázi byly dva různé typy sýrů zaočkovány S. aureus za účelem simulace sekundární kontaminace při výrobě sýrů. Vzorky byly odebírány v průběhu 4 týdnů. Kritické faktory jako jsou kompetitivní mikrofóra nebo pH, které jsou zodpovědné za regulaci virulence S. aureus byly sledovány. Snažili jsem se rozlišit situace při kterých: (i) není pozorován růst, ale objevuje se produkce enterotoxinu a (ii) dochází k růstu ale bez produkce enterotoxinu.
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Vliv PUFA n-3 na expresi genů kódujících proteiny řídící homeostázu cholesterolu
Hyblerová, Dagmar
The aim of this study was to confirm that the polyunsaturated fatty acids n-3 (n-3 PUFA) have a positive effect on plasma lipids. These acids can reduce cholesterol by increasing gene expression Insig-1 while decreasing the expression of genes encoding Hmgcr and Ldlr. We tested in experimental rats, which were added to the feed mixture of 6 % safflower oil , 6 % fish oil or 6 % of the oil from the algae Schizochytrium. Relative gene expression was Insig-1 in the test group with addition of fish oil to 120% of controls (P<0.05) and in the group with addition of oils from algae Schizochytrium the relative expression of 170 % of control (P<0.05). These results confirm our hypothesis, only a part, as the relative expression of the gene and Hmgcr and Ldlr was in the test group with addition of fish oil 103% (P>0.05) and 101 % of control (P>0.05) and in the group with addition of oils from algae Schizochytrium the relative expression of 117% (P>0.05) and 156 % (P>0.05) compared to control. Thus, to reduce the relative expression of these genes did not. However, we have shown that n-3 PUFA contribute to a reduction in plasma cholesterol and in this case up to 20 % of control. The concentration of cholesterol in the group with addition of safflower oil was 1.35 mmol.l-1, the group with the addition of fish oil 0.98 mmol.l-1.
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The application off molecular biological methods in diagnosing the cythomegalovirus in the clinical matherials
ŠIMEROVÁ, Erika
Human cytomegalovirus (HCMV or CMV) is a human viral pathogen from a group of herpetic viruses. Isolation of DNA can be done manually or with the automatic machines - isolators. In my work I concentrated to compare these two different isolations. I did manual isolation with the help of impervious QIAmp DNA Mini Kitu made by QIAGEN and automatic isolation with isolator MagCore using MagCore Genomic whole Blood Kit (RBCBioscience). To compare the isolations blood and plasma were used as clinical materials and their origin was the same. Both methods were used afterwards to set up the qualitative and comparative CMV DNA. The result means that both isolations are comparable - both can be used with the same quantitative result. In this work there is also compared the qualitative determination DNA CMV by using two PCR methods. One of them was the "In house" method in which are two sections of viral DNA CMV ampificated by the couples of specific primers and later there are detected by the agarose gel. Visualisation of these products on agarose gel is going on with the help of intercalatial reagent - ethidium bromide. This method is very sensible but it takes a lot of time.
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Leiden mutation, its signification and methods of examination
FIALOVÁ, Kateřina
The subject of this thesis is Leiden mutation, its signification and methods of examination. This work deals with frequency of occurrence F V Leiden mutation in samples of examinants in the centre of laboratory medicine BioLab, spol. s. r. o., in Klatovy. Thrombophilia (hypercoagulation) is a genetic or acquired failure of hemostatic mechanistic system, which is connected with increased risk of the occurrence of thrombosis. The most important and the most frequent effect of thrombophilia is the venous thromboembolism. This concept means both deep venous thrombosis and/or pulmonary embolism and their development is always caused by several factors which are genetic or acquired. Mutation of factor V was described in 1993 as the resistance against activated protein (so called APC-resistance). It occurs most commonly as a genetic thrombophilia of white race where it can be found in 40% of patients with diagnosed thromboembolism. Leiden mutation in a gene for factor V is the most frequent predisposition for thrombosis. The risk of development of venous thromboembolism differs according as the fact if it is a heterozygotic or homozygotic carrier. Heterozygotic individuals are 7 times more riskly as for the development of venous thrombosis while the risk level in homozygotic individuals is 80 times. The rate of prevalence of FV Leiden mutation in the world ranges from 0 to 15 %. APC- resistance is caused by point mutation in the polynucleotide chain of a gene for factor V. Here it concerns the exchange of the nucleotide sequence respectively nitrogen basis of guanine and adenine for the position 1691 (G1691A). It creates a triplet which codes in position 506 in the place of amino acid arginine -Arg, R, glutamine -Gln, Q(R506Q). It causes resistance of the active factor V against protein C, which is to inactivate it. The factor V stays henceforth pro- coagulative and thus the creation of thrombin increases together with the risk of thromboembolic disease. The goal of this thesis was summarising of research findings related to the focused theme Leiden mutation, its signification and methods of examination, analyze whole blood samples of patients examined at Leiden mutation carrier status in the center of Laboratory Medicine BioLab, spol. s. r. o. in Klatovy and in consequence and then evaluate and interpret the results of the found out results. Carrier of FV Leiden mutation was observed in a group of patients of both sexes coming from Plzeň and Ústí nad Labem region using real-time PCR. Of the 169 patients examined 111 samples were obtained from women and 58 samples from men. Among women there were found 21 heterozygous carriers (18.9%) and 1 homozygous carrier of (0.9%) FV Leiden mutation. The male population was observed incidence of 17 heterozygotes (29.3%). None of the men did not hold the homozygous genotype FV Leiden mutation. Although the men were twice as frequent carriers of that mutation was not statistical analyzes demonstrated the effect of gender on the transfer FV Leiden mutation (P = 0.2).
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Comparison of methods for determination of Neisseria meningitidis in Nemocnice ČB a.s.
SKOŘEPOVÁ, Lucie
Molecular biology methods are of great importance for routine diagnostics of infections diseases. In this work two such methods were compared for the case of diagnostics of Neisseria meningitides in Nemocnice ČB a.s. Nemocnice ČB a.s. is a municipal hospital owned by South Bohemia Region. I hypothesised that real-time polymerase chain reaction provides lower analysis time compared to nested polymerase chain reaction. These two methods are routinely used in Central Laboratories of the hospital and it is feasible to use both of them for the diagnostics of Neisseria meningitides. During the investigation I have found another method useful for the diagnostics of this pathogen. Latex agglutination test is an immunological reaction used to detect antibodies which appear as an immune response to infection. In spite of very quick output, the immunological origin of the latex agglutination test brings serious limitations: any suppression of the immune response or degradation of the antibodies leads to false negative results of the test. Lower reliability of the latex agglutination test compared to various polymerase chain reactions based on the detection of bacterial DNA strengthen my devotion to compare the two methodologies of reliable polymerase chain reaction and led to a decision not to include the latex agglutination test into the original research plane. In good agreement with the original expectation, practical evaluation of tests based on real-time polymerase chain reaction and on nested polymerase chain reaction displayed significant differences in the analysis time (3 h 40 min versus 7 h 30 min respectively). Another advantage of the test based on real-time polymerase chain reaction is its higher sensitivity of one order of magnitude more than in the test based on nested polymerase chain.
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PCR based assay for detection garden pea lec gene
Vráblík, Aleš ; Hodek, Jan ; Ovesná, Jaroslava
The method was developed and verified by staff of National reference laboratory for GMO identification and DNA fingerprinting suited for governmental control laboratories and private laboratories that analyze food and feed derived from various plant matrices. Method can be applied for quantification of GM pea in a sample, when reference gene is used as a standard. The method describe internal garden pea specific gene, lektin in this case, detection using PCR and real-time PCR. The general principle of the assay relies on lektin specific sequence presence that represents a species specific gene of garden pea. In many matrices DNA is damaged so amplification of short stretches of DNA is used. After amplification of target exploiting newly developer species specific gene primers PCR products are separated by electrophoresis in agarose gel. Resulting band is visualized by UV light and its position is compare with size standard. Alternatively, real-time PCR and ABI platform in combination with SYBR® Green can be used. Reaction parameters are described and specificity of reaction was verified. LOD as well as LOQ was defined.
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