Nuts of all kinds are common ingredients in food. For nut allergy sufferers the frequent use of nuts cause problems and "hidden" nuts in food products may elicit allergic reaction when such foods are consumed. Methods for detecting and quantifying walnut (and other nuts) with high sensitivity and specificity are therefore very important. The objective of this project was to verify the specificity of a rabbit antiserum against walnut with immunodiffusion and to determine the size of the dominant walnut antigens with Western blotting. In addition, a commercial sandwich ELISA for walnut quantification was validated and compared with a qualitative real-time PCR. The rabbit antiserum proved to be less specific but after absorption with cross-reacting nuts and seeds it showed high specificity. The ELISA kit reacted, except for walnut, with pecan and slightly with other nuts and seeds tested. The PCR showed an absolute specificity to walnut. As low levels as 2.5mg walnut/kg can be quantified with the ELISA. This is 8 to 100 fold less than with the PCR method. It is therefore concluded that the ELISA kit is more sensitive than the PCR method but the PCR method is more specific than the ELISA kit.

The project aim is to determine if good quality DNA can be extracted from barley, malt and hop, ingredients used in beer brewing. Good quality DNA is important in DNA fingerprinting techniques which can be used for identification of ingredients. The 3 methods tested are the cetyltrimethylammonium bromide (CTAB) method, QIAGEN DNeasy Plant Mini Kit and Meyer’s method as published in 1996 with QIAGEN DNeasy Plant Mini Kit in combination. To evaluate the DNA quality after extraction we used 3 different techniques: (i) spectrophotometry to estimate purity by using the ratio A260/A280; (ii) agarose gel electrophoresis after DNA extraction to determine the success of the extraction and evaluate the amount of high molecular weight DNA and degradation; and (iii) the polymerase chain reaction with 4 different primer pairs, together with agarose gel electrophoresis, to determine if the extracted DNA could be used in downstream applications, see the effect of inhibitors and estimate the fragmentisation of the DNA. The results achieved using the above mentioned methods were then used to evaluate the success of each of the extraction methods in their function of extracting high quality DNA from barley, malt and hop as well as determining whether the treatment of the ingredients has an effect on the DNA quality.

Photosensitizing molecules and/or metal complexes that interact with DNA via intercalation and groove binding have potential applications as molecular structural probes, as footprinting reagents and in photodynamic therapeutics. To this regard, small molecules that bind to DNA and the energetics involved in these interactions, acridine-based therapeutics, photosensitization, photodynamic therapy, phenothiazine-mediated photosensitization, DNA photocleavage reaction mechanisms and photosensitizing metal complexes are introduced in Chapter I. Next, in Chapter II, the synthesis of a photonuclease consisting of a 3,6-acridinediamine chromophore attached to four metal-coordinating imidazole rings is described. The DNA photocleavage yields, emission quantum yields, and thermal denaturation studies by this acridine-imadazole conjugate in the presence of 16 metal salts are also reported. In Chapter III is the synthesis of a bisacridine covalently tethered to a copper(II)-binding pyridine linker. Additionally, DNA photocleavage studies as well as DNA binding affinity and binding mode(s) of this bisacridine incorporating the copper(II)-binding pyridine linker are examined. The syntheses, characterization, DNA photocleavage studies, DNA thermal denaturation, and viscometric measurements of three new phenothiazinium photosensitizers are described in Chapters IV and V. Collectively, markedly enhanced DNA photocleavage yields are observed in the presence of metals (Chapters II-III) or in comparison to a parent molecule, Chapters II and IV. DNA melting isotherms show higher levels of duplex stabilization with the acridines, specifically in the presence of several metals (Chapter II-III) as well as with the phenothiazine-based ligands (Chapters IV-V). Moreover, different DNA binding modes were observed depending on metal complexation (Chapter III) and nucleic acid structure (Chapter IV). Finally, Chapter VI describes a small project implemented as a National Science Foundation pedagogical laboratory exercise in which a non-invasive procedure for DNA isolation from human cheek cells was utilized with the polymerase chain reaction to amplify alleles encoding a single nucleotide polymorphism involved in normal human color vision.

Early detection of infection, identification of microorganism, and correct choice of antibiotics are critical in the management of sepsis. Quantitative real-time polymerase chain reaction (RT-PCR) has the potential to improve the timeliness, sensitivity, and accuracy of detecting pathogens. In this study we utilize this method to detect the enteric bacterial counts in the blood from patients with systemic inflammatory response syndrome (SIRS) in the emergency department (ED). The universal primers utilized in RT-PCR are specific for 23S ribosomal DNA (rDNA) and wec F gene. The results show that in SIRS patients with positive culture results from specimen collected within 10 days after presenting to ED, and patients surviving for less than 28 days, the serum bacterial DNA load of enteric Gram negative bacilli is higher. In SIRS patients with shock, patients fulfilling both white blood cell counts and respiratory criteria of SIRS, and patients fulfilling both white blood cell counts and respiratory criteria of SIRS with Acute Physiology and Chronic Health Evaluation II score more than 20, the serum bacterial DNA load of enteric Gram negative bacilli and 28-day mortality are both higher. These results suggest that bacterial translocation may happen in patients with SIRS and may be related to higher mortality in patients with SIRS.

Canine babesiosis is an important worldwide disease caused by protozoan hemoparasites of the genus Babesia, which are primarily transmitted to a dog by the bite of an Ixodid tick, although vertical transmission has recently been reported. The disease is typically characterized by hemolytic anemia, fever, splenomegaly, and thrombocytopenia, with clinical signs ranging from clinically normal to acute anemia. Death may even result in some severe cases. Two species of Babesia, Babesia gibsoni and Babesia canis, have long been known to cause babesiosis in dogs. To date, almost all B. gibsoni infections in the United States have been reported in American Pit Bull Terriers or in dogs associated with the breed through either transfusion or fighting. Dog blood samples received from kennels, shelters, and veterinary clinics throughout Texas were tested for the presence of B. gibsoni and B. canis. A total of 254 samples were tested for B. gibsoni and B. canis by light microscopy and polymerase chain reaction (PCR). Babesia gibsoni was detected in four of the dogs tested and B. canis was detected in one of the dogs tested. The average packed cell volumes (PCVs) of infected dogs were compared with those of uninfected dogs, with the infected, on average, having lower PCVs. Molecular characterization of the small subunit ribosomal RNA gene and the ribosomal RNA internal transcribed spacer regions was performed on all sequences obtained in this study, and results were consistent with those previously reported for B. gibsoni and B. canis. Also, positive samples and additional samples provided by North Carolinia State University were used to initiate in vitro cultures of the parasites. To date, one isolate of a large unknown Babesia sp. from a North Carolina dog was successfully established in vitro. The establishment of Babesia spp. parasites in culture may aid in the development of a vaccine for babesiosis and will also be beneficial in improving diagnostic tests for the parasite.

Exons are expression DNA sequences. A DNA sequence which includes gene has exons and introns. During transcription and translation, introns will be removed, and exons will remain to become protein. Many researchers need exon primers for PCR experiments. However, it is a difficult to find that many exon primers satisfy all primer design constraints at the same time. Here, we proposed an efficient exon primer design algorithm. The algorithm applies multiobjective genetic algorithm (MGA) instead of the single objective algorithm which can easily lend to unsuitable solutions. And a hash-index algorithm is applied to make specificity checking in a reasonable time. The algorithm has tested by a variety of mRNA sequences. These dry dock experiments show that our proposed algorithm can find primers which satisfy all exon primer design constraints.

No description available.

In polymerase chain reaction (PCR), in order to amplify massive DNA sequences successfully, it needs to design an appropriate primer pair. The constraints derived from the traits of PCR for proceeding PCR are used in searching for primer pairs. In this paper, in order to decrease the searching space and to increase the feasible quality of primers, a double orthogonal arrays intelligent crossover genetic algorithm (DOAIGA) is used to solve the primer design problem. DOAIGA combines the traditional genetic algorithm and the Taguchi methodology to efficiently search feasible primers under required constraints. The proposed intelligent crossover subsystem mainly concentrates on the better genes more systematic. The key point of DOAIGA is to achieve the elitism goal by applying the orthogonal arrays (OAs) that is used in quality engineering with a small amount of experiment features. In this thesis, the double orthogonal arrays are used to approach a better forward and reverse primers separately. Compared to the current existing softwares, DOAIGA can obtain feasible primer pairs more effectively. Finally the correctness of primer pair is verified by PCR experiment.

Tez (Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Enfeksiyon Hastalıkları ve Klinik Mikrobiyoloji Anabilim Dalı, 2008. / Kaynakça var.

Tez (Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Biyokimya Anabilim Dalı, 2008. / Kaynakça var.