|
|
The use of magnetic microparticles for bacterial DNA isolation
Hrudíková, Radka ; Horák, Daniel (referee) ; Španová, Alena (advisor)
The aim of the work was testing of two types of magnetic mikrosheres functionalised with –COOH groups for the isolation of bacterial DNA. Isolation of DNA was carried out from crude lysates of cells prepared from pure culture of Lactobacillus paracassei RL-10 in the presence of binding buffer with 2 M NaCl and 16% PEG 6000. The influence of RNA degradation by enzyme RNase A on the amount of isolated DNA was investigated. It was estimated that RNA degradation affects the amount of DNA isolated. The amount of DNA depended on the type of microparticles. Higher amounts of DNA were isolated using particles with higher content of carboxyl groups. DNA applicable in PCR was isolated using both types of microsheres. In next part of the work, microparticles functionalised with –NH2 groups were used to DNA isolation using electrostatic forces. It was shown that buffer with lower pH is suitable for DNA adsorption onto magnetic microparticles.
|
|
|
Identification of lactic acid bacteria in hard cheeses using amplification methods
Herzogová, Jitka ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Diploma thesis was focused on identification of lactic acid bacteria of species Lactococcus lactis and subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris using species and subspecies specific polymerace chain reaction (PCR). PCR method was used for identification of bacteria of species Lactococcus lactis in 10 samples of hard cheeses. The method of sample preparation was evaluated for hard cheeses with the aim to receive sufficient amount of cells for the preparation of crude cell lysates. Whole DNA in quality suitable for PCR was separated using magnetic microspheres P(HEMA-co-GMA) in the presence of polyethylenglycol (PEG 6000) and sodium chloride. DNA isolated by phenol extraction was used as control of DNA isolation. PCR was used to the analysis of 7 strains of Lactococcus lactis from Collection of dairy microorganisms Laktofora (CCDM). Altogether 5 or 2 strains were identified into subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris, respectively.
|
|
|
The application of magnetic particles for DNA isolation from cereal products
Starenkova, Anastasiia ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The thesis has been focused on micro method for isolation of PCR- ready DNA iusing magnetic particles from cereal products. Cereal biscuits and cereal products for babies were selected for the analysis. These were homogenized using plastic copist in lysis buffer with cetyltrimethylammonium bromide (CTAB). The homogenates were purified using chloroform- octanol mixture. The effect of isopropanol in the preparation of homogenates was tested, too. Homogenates were used for DNA isolation by magnetic particles. Two ways to isolate magnetic particles with bounded DNA (magnetic separator and magnetic needle have been tested. Isolated DNA was analyzed spectrophotometrically its concentration and purity were assessed. . After that, amplification of the DNA was tested in PCR. Two sets of primers specific for plant ribosomal DNA were used for their amplification. PCR products of expected length 700 bp and 220 bp were detected by agarose gel electrophoresis. It was shown that DNA isolated from seeds and cereal products using magnetic particles was in PCR-ready quality.
|
|
|
Detekce patogenů lilku bramboru přenosných sadbou
Bačová, Nikola
This bachelor's thesis focuses on seed-borne pathogens of potatoes and methods for determining these pathogens through polymerase chain reaction (PCR). The thesis describes the methods of detection of Rhizoctonia solani and Helminthosporium solani pathogens. Both pathogens, Rhizoctonia solani and Helminthosporium solani, are important pathogens of potatoes, affecting the appearance and marketability of the tubers and at the same time limiting the use of the infected seedlings. For the Rhizoctonia solani pathogen, the detection and quantification methodology was optimized using real-time polymerase chain reaction (qPCR) using ARSF4/R4 primers, and for the Helminthosporium solani pathogen, the qPCR detection methodology was optimized using the Hs1NF1/Hs2NR1 primers.
|
|
|
Detekce patogenů lilku bramboru přežívajících v půdě
Valkovičová, Nikola
The bachelor's thesis dealt with the detection and sequencing of soil-borne pathogens of pota-to eggplant. The focus is mainly on pathogens of the genus Fusarium. These pathogens cause such potato rot, which forms white mycelium coatings and subsequently mummifies, that it is not suitable for consumption and the yield is reduced. One of the most accurate methods was chosen for the detection of pathogens – PCR. For the detection of Fusarium pathogens from other potato tubers, conventional PCR was used with non-specific primers ITS4 and ITS5 annealing to the region between the genes that code for ITS4 and ITS5 RNA. For the detection of pathogens from the soil, real–time PCR was chosen with primers ITS1F and AFP346 sitting in the region that encode nRNA and space bars ITS1 and ITS2. The tested soil was artificially inoculated with an unknown isolate of the genus Fusarium obtained from a potato tuber and a collection isolate of Fusarium solani var. coeruleum. Se-quencing confirmed infection of the potato tuber with Fusarium culmorum, clarifying the dif-ficulty of quantification.
|
|
|
Influence of matrix type on the authentication of foodstuffs containing fruits
Kopková, Pavlína ; Strečanská, Paulína (referee) ; Fialová, Lenka (advisor)
Certain types of food, mainly the more expensive ones, are often adulterated to reduce their manufacturing price. However, this reduces their quality and can also have a negative impact on the health of the consumer. Children's fruit products are also targeted by fraudulent producers, where the declared fruit is most often replaced by a cheaper version. This work focuses on the detection of adulterated foods using various analytical methods, in particular PCR. The theoretical part focuses on the issue of food adulteration, the analytical methods used for detecting adulteration, and also on mango and banana which are determined in this work. The aim of this thesis was to determine what effect the type of matrix has on the determination of fruit components in food by PCR. Three types of matrix were used for this purpose - fruit puree, smoothie, and bars. An important task was to optimize the DNA isolation to achieve adequate purity and concentration of DNA. Then, the amplifiability of the obtained DNA was verified. The DNA isolates were then analyzed by multiplex PCR with primers specific for mango and banana. The results were verified by agarose gel electrophoresis. Subsequently, it was possible to determine that the fruit component in bars and fresh smoothies was the most easily analyzed by PCR and, on the contrary, the determination was problematic for puree. The instrumental part was focused on the determination of phenolic compounds in the products by HPLC. For this purpose, optimization of the extraction of phenolic compounds was necessary. This method was able to detect the presence of mangoes in all samples.
|
|
|
Cytomegalovirus infection in transplant patients
Dvořák, Jan ; Tachezy, Ruth (advisor) ; Harant, Karel (referee)
Cytomegalovirus (HCMV) is a ubiquitous human -herpesvirus highly prevalent in the population. HCMV is transmitted by close contact between individuals. In infected person this virus causes mainly asymptomatic primary infection, after which the latency is established. In pregnant women HCMV infection can lead to abortions, defects of the fetus and congenital abnormalities of newborn babies. Even more serious complications are caused by this virus in the immunocompromised patients, especially those infected by HIV and in patients who undergo solid organ transplantation and hematopoietic stem cell transplantation. This work is a complex report about HCMV biology with emphasis on complications which HCMV causes in patients after solid organ transplantation and hematopoietic stem cell transplantation. This article also contains summary of the methods used for diagnostic of HCMV infection and monitoring and prevention of HCMV associated diseases. Keywords: Cytomegalovirus, hematopoietic stem cell transplantation, solid organ transplantation, detection, monitoring, polymerase chain reaction, cellular immunity, humoral immunity
|
|
|
Detection of chimeras in amplicon sequencing
Heřmánková, Kristýna ; Jurečková, Kateřina (referee) ; Sedlář, Karel (advisor)
Chimeric sequences are the most common artifacts that can occur in sequencing data after the sample amplification using the polymerase chain reaction. The presence of these artifacts can negatively affect results of the analysis. Therefore, the detection and subsequent filtration of chimeric sequences is an important step in the computational processing of sequencing data. This work deals with the principle of chimera formation and the possibility of reducing their occurrence. The aim of this work is to implement an algorithm for chimeras detection in R language and testing its accuracy on data provided by the Veterinary Research Institute in Brno.
|
|
|
Laboratory diagnostics of micrometastases in breast cancer patients
Mikulová, Veronika ; Zima, Tomáš (advisor) ; Svoboda, Marek (referee) ; Průša, Richard (referee)
Introduction: The presence of circulating tumor cells (CTC) in the peripheral blood has been associated with worse prognosis and early relapse in breast cancer patients. CTC determination in the peripheral blood has been considered as a liquid biopsy. The aim of this project was to analyze the presence of CTC followed by their molecular characterization with the potential use not only as a new biomarker for real-time monitoring of therapy efficacy but also as a suitable tool for patient's stratification and individualization of treatment for breast cancer. Methods: A total of 54 patients with diagnosed early breast cancer were enrolled into a prospective study. Ten millilitres of peripheral blood were sequentially collected to test for the presence and characterization of CTC during the follow-up of patients. CTC isolation and detection was performed by AdnaTest BreastCancer™ (AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC- lysates. cDNA from isolated CTC has been further used for newly optimized qPCR assays for breast tumor and therapy resistance associated genes: TOP1, TOP2A, CSTD, ST6GAL, KRT19 and reference gene actin. qPCR results have been analyzed by Genex software (MultiD Analysis). Results: 195 blood samples have been...
|
|
|
Methods for storing and archiving zoological tissue collections, and identification based on DNA barcoding
Aghová, Tatiana ; Benda, Petr ; Brejcha, Jindřich ; Dolejš, Petr ; Kyralová, Eva ; Mlíkovský, Jiří ; Moravec,Jiří ; Šanda, Radek ; Štundl, Jan ; Tkoč, Michal ; Vondráček, Dominik
Fulltext:  PDF; PDF; PDF; PDF
|
guest ::
