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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Development Of A Sandwich-type Dna Array Platform For The Detection Of Label-free Oligonucleotide Targets

Cansiz, Sena 01 October 2010 (has links) (PDF)
DNA arrays have become a major bioanalytical method as they enable high-throughput screening and they can be manufactured on different surfaces depending on the nature of diagnostic purpose. However, current technologies to produce and detect arrays of DNA probes are expensive due to the requirement of specialized instrumentation. In this study we have established an array platform in sandwich hybridization format for the detection of label-free nucleic acid targets. Unlike direct hybridization, which is the main microarray hybridization principle, sandwich assay enables unlabeled target detection, lowering the cost and assay time. To this end, sequence specific signal development was achieved by a sandwich complex which is composed of a surface immobilized capture DNA probe (Probe1) and a fluorescein-tagged signal DNA probe (Probe 2), which are partially complementary to the sequence to be analyzed (target oligonucleotide). As the solid support of the array platform both 3-aminopropyl-3-methoxysilane (APTMS) activated and commercially purchased poly-L lysine coated glass slides were used and due to the less background noise property the latter one was preferred. Similarly, for the immobilization of the capture Probe (P1) onto the solid support two different methods were tried / heat immobilization and immobilization via a heterobifunctional cross-linker (HBCL). In regard to the experiments, it is observed that using a cross-linker instead of heat immobilization reduces the ratio of false negative control results in a significant manner. Following the solid support and immobilization method choice comparative optimization studies which include cross-linker type, probe concentration, sensitivity of the platform and hybridization conditions (sequence, temperature and duration) were conducted. Optimum hybridization signal was obtained with a 32.5
2

Development Of An Oligonucleotide Based Sandwich Array Platform For The Detection Of Transgenic Elements From Plant Sources Using Label-free Pcr Products

Gul, Fatma 01 October 2010 (has links) (PDF)
Advances in DNA micro and macroarray technologies made these high-throughput systems good candidates for the development of cheaper, faster and easier qualitative and quantitative detection methods. In this study, a simple and cost effective sandwich hybridization-based method has been developed for the rapid and sensitive detection of various unmodified recombinant elements in transgenic plants. Attention was first focused on the optimization of conditions such as time, concentration and temperature using commercial ssDNA, which in turn could be used for real sample detection. In this sandwich-type DNA chip platform, capture probes complementary to the first half of recombinant element (target adapter) were immobilized onto poly-L-lysine covered conventional microscope slides. PCR-amplified un-purified target adapter and biotin labeled detection probe, which is complementary to the second half of target adapter, were hybridized in solution-phase to complementary capture probes to create a sandwiched tripartite complex. Later, hybridization signal was visualized by the attachment of streptavidin conjugated Quantum Dot to the sandwiched complex under UV illumination. Sandwich based array system that has been developed in this study allows multiplex screening of GMO events on a single DNA chip platform. 35S promoter, NOS terminator, CRY1Ab and BAR target sequences were successfully detected on the same DNA chip platform. The platform was able to detect unlabeled PCR amplified DNA fragments of CaMV 35S promoter sequence and NOS terminator and BAR transgene sequences from transgenic potato plants and NK603 Certified GMO Reference material, respectively. The DNA-chip platform developed in this study will allow multiple detection of label-free PCR-amplified transgenic elements from real GMO samples on a single slide via a cost effective, fast, reliable and sensitive sandwich hybridization assay.
3

Improved enrichment cultivation of selected food-contaminating bacteria

Taskila, S. (Sanna) 16 November 2010 (has links)
Abstract The aim of this work was to assess and improve the enrichment cultivation of food-contaminating bacteria prior to detection by means of RNA-based sandwich hybridization assay (SHA). The examples of beer-spoiling lactic acid bacteria (LAB) and food-borne Salmonella Typhimurium were selected based on their relevance in Finnish food industry. Also universal challenges affecting on the selection of the enrichment cultivation procedure are discussed, including some potential possibilities for improved enrichment cultivation. The results of this study may therefore be used for the assessment of the efficiency of bacterial cultivation in other applications. The evaluation of the enrichment cultivation procedures prior to SHA lead to following conclusions: i) the enrichment cultivation procedure is necessary prior to rRNA-based SHA, and it directly influences the accuracy of SHA; ii) the improvement of the enrichment cultivation may allow faster recovery and growth of bacteria; iii) the improved recovery of bacteria can be achieved by reducing environmental stress factors in the enrichment culture; and iv) the growth of bacteria may be accelerated by assuring the selectivity of medium and allowing accessibility to growth factors. Several growth factors were studied by means of full factorial design and response surface modeling. Measured cell densities, as well as predicted lag-times and maximum growth rates in the bacterial cultures were used as responses. The results show that small shifts in the cultivation conditions extend the lag-time and decrease the growth rate of both LAB and Salmonella. Besides adjusting the temperature and pH, the growth of LAB was facilitated by reducing osmotic and oxidative stresses in the enrichment medium. In this study, a novel enzyme controlled glucose delivery system was used for the first time in the enrichment cultivation of food-contaminating bacteria. The glucose delivery system improved the growth of LAB in single strain cultures and in actual brewing process samples. The recovery of injured Salmonella was also enhanced by using the glucose delivery system together with selective siderophore ferrioxamine E, both in terms of reduced lag-times and increased growth rates. Based on the SHA, the adjusted BPW broth enhanced the molecular detection of heat-injured Salmonella in meat.
4

Detection of harmful microbes and their metabolites with novel methods in the agri-food production chain

Nieminen, T. (Timo) 12 January 2009 (has links)
Abstract This thesis aimed at developing methods for tracking the environmental origins of microbial contaminants of the food chain. We worked on three targets: i) environmental mycobacteria ii) toxinogenic Bacillus species iii) post-harvest fungi in strawberry jam. Our aim was to develop methods for early detection of the above contaminants, which have the potential to endanger consumer health. We developed a novel method based on 16S rRNA hybridization for tracking the reservoirs of potentially pathogenic environmental mycobacteria in piggeries and soil. From 1010 to 1012 16S rRNA molecules of environmental mycobacteria were found per gram of peat, wood shavings and straw in piggeries with a high prevalence of infections. These beddings may thus be a source of mycobacteria for pigs. We found 1010–1011 of mycobacterial 16S rRNA molecules per gram of Finnish forest soil, indicating that the soil contained 107–109 mycobacteria per gram. These numbers exceed the previous cultivation-based estimates of mycobacterial content in Finnish soils. To elucidate the role of mastitis in the input of toxinogenic Bacillus into the dairy production chain, milks were sampled from mastitic cows. Twenty-three Bacillus isolates were screened for toxins using the sperm cell motility inhibition assay. Four of the six toxinogenic isolates found were identified as Bacillus pumilus and two as Bacillus licheniformis. The isolates produced toxic substances that were heat-stable (100 °C) and soluble in methanol, thus being of non-protein nature. The extracts prepared from the toxin-producing isolates disrupted the plasma membrane of exposed sperm cells at concentrations 1–15 μg ml-1 (B. pumilus) 20–30 μg ml-1 (B. licheniformis). The toxic action of the mastitis-associated B. licheniformis strains was similar to that of the lipopeptide lichenysin A. The genes for lichenysin synthetase were found in these strains by PCR. This study revealed that heat-stable toxin-producing strains of B. pumilus and B. licheniformis occur in milk of mastitic milking cows. They may enter the dairy production chain when milk of clinically healthy cows recovered from mastitis is sent to dairies. Many foodborne contaminant fungi are known to produce volatile organic compounds. We investigated the suitability of such metabolites as early indicators of fungal contamination of strawberry jam. We found that volatile organic compounds commonly produced by the contaminant fungi in strawberry jam were 2-pentanone, styrene, 3-methyl-1-butanol, 1,3-pentadiene and ethanol. The results indicate that these compounds could be used to detect fungal contamination of jam.

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