Quantitative real-time PCR (QPCR) technology has been very useful for diagnosis of viral diseases. QPCR has recently reached a level of sensitivity, simplicity, and reproducibility which allows a large number of samples to be screened rapidly, make it a suitable tool for the clinical virology diagnostics. In this thesis, broadly targeted and degenerated quantitative QPCR assays were used. A somewhat novel single-tube real-time reverse transcription-polymerase chain reaction (QRT-PCR), with takes advantage of ability of rTth DNA polymerase to reverse transcribe RNA in the presence of Mn2+ at elevated temperatures and includes protection against amplimer contamination by using thermolabile UNG, was developed. A new technique for diagnostic of recent viral infection by detection of viral immunoglobulin M (IgM) was also developed. In the first paper, a sensitive single-tube QRT-PCR for detection of enteroviral RNA in patients with aseptic meningitis was presented. In the second paper, a single-serum-dilution real-time PCR-based PIA (PCR-enhanced immunoassay), called quantitative PIA (QPIA), to detect enterovirus IgM for diagnosis of EV infection in patients with aseptic meningitis, was also developed. In the third paper, a broadly targeted, simple, single tube degenerated quantitative QPCR technique for detection of JCV, BKV and SV40 DNA was developed. A conserved region of the VP2 gene of JCV, BKV and SV40 was targeted. A false positive result due to contamination with commonly used SV40 T-antigen plasmids was therefore avoided. In manuscript four, the QPIA assay provide a rational strategy for detection of EV IgM, allows the use of viral antigens isolate from newly diagnosed Type 1 diabetes patients (T1D-EV-QPIA) to measured IgM against diabetogenic viruses in serum from newly diagnosed T1D children, siblings, and healthy children. To conclude, novel broadly targeted real-time PCR methods for diagnosis of entero- and polyoma viral infections were developed.

Il tema principale di questa tesi di dottorato verte sul metabolismo secondario di F. verticillioides, con particolare interesse alla via biosintetica, produzione e mascheramento delle fumonisine B (FB), fumonisine A (FA), fumonisine C (FC) e bikaverina, studiati in relazione all’ecologia fungina. E’ stato osservato che l’acqua libera (aw) esercita un effetto significativo maggiore rispetto alla temperatura sul metabolismo secondario di F. verticillioides. Alti valori di aw favoriscono l’espressione dei geni FUM ed una maggiore sintesi delle FB, FA e FC. Inoltre la produzione di bikaverina e l’espressione di BIK1 sono influenzate dall’ aw nello stesso modo che la produzione di FB e l’espressione dei geni FUM. Anche il tempo di incubazione è un fattore critico per la produzione di FB in F. verticillioides, così come per F. proliferatum: la produzione di FB, FA e FC aumenta nel tempo fino a 30 giorni, periodo dopo il quale si notano differenze tra le due specie fungine. Riguardo le fumonisine nascoste, queste sono state ritrovate in colture sia di F. verticillioides che di F. proliferatum. Non è stata registrata sintesi alcuna in colture cresciute su substrato di crescita sintetico, ma al contrario solo su colture cresciute su substrato a base di mais. / The main topic of this PhD thesis is F. verticillioides secondary metabolism, with regard to fumonisin B (FB), fumonisin A (FA), fumonisin C (FC) and bikaverin biosynthetic pathways and production and masking, studied in relation to fungal ecology. What we found is that water activity (aw) has a more significant effect than temperature on F. verticillioides secondary metabolism. Moreover bikaverin production and BIK1 expression are influenced by aw in the same way as FB production and FUM gene expression respectively. High aw levels favour FUM gene expression and allow the highest synthesis of FB, FA and FC. Incubation time is also critical for FB production both for F. verticillioides and F. proliferatum: the general trend is that FB, FA and FC production increases with time, up to 30 days, period after which differences can be noticed between F. verticillioides and F. proliferatum. As regard masked fumonisins, they are recovered in F. verticillioides and also F. proliferatum cultures, furthermore no synthesis is observed on cultures grown on synthetic medium, but only in those grown on maize-based substrates.

Food allergies are an important health problem and affect up to 2% of the adult population and 8% of children worldwide. Under the Food Allergen Labeling and Consumer Protection Act (FALCPA) of 2004, foods that contain or derive from the "Big 8" allergens (milk, egg, finfish, crustacean shellfish, tree nuts, peanuts, wheat, and soybeans) must be declared and the "common or usual name" of the allergen source must be printed on the label of the food product. Currently, the most common used detection methods for food allergens are enzyme-linked immunosorbent assay (ELISA) based. ELISA, a protein-based method, targets specific allergen(s) and detects by colorimetric reaction following binding with a specific-enzyme labeled antibody. However, studies have demonstrated that matrix interference and heat treatment can interfere with the detectability of commercial ELISA kits. An alternative approach to targeting the allergen in soy is to use deoxyribonucleic acid (DNA) as a unique marker that can be used to indicate the presence of soy in food. According to FALCPA the source of an allergen should be declared on the label, therefore identifying an allergen, such as soy, by DNA detection could be a valid means of meeting FALCPA requirements. Real-time polymerase chain reaction (real-time PCR), a DNA-based method, can identify the presence of soy through amplification of specific sequences of DNA through the use of primers. However, the sensitivity of real-time PCR can be influenced by the amplification protocol, primer design and DNA extraction methods. Thus, the main objectives of this study were to 1) verify the specificity of primers designed to detect soy DNA from different soy products, 2) optimize the previously developed real-time PCR protocol to detect soy DNA, 3) investigate the application of two commercially available DNA recovery systems (column and magnetic beads) to recover soy DNA from different forms of soy products using real-time PCR and 4) determine the effect of food matrices and thermal processing on soy detection using DNA and ELISA methods. In this study, Wizard Magnetic DNA Purification system kit (Promega, Madison, WI) was selected as the column DNA recovery system while DNeasy mericon Food Kit (Qiagen, Valencia, CA) was selected as the magnetic beads system. Neogen Veratox for soy allergen was selected as the ELISA system. The evaluations of both DNA recovery systems were conducted on soy protein isolates (SPI), powdered soybean and soymilk. The effect of thermal processing in soy detection was conducted on four different food matrices (protein, fat, carbohydrate and water). Each food matrix was spiked with 10% soy protein isolates and heated at 95ºC for an hour. Both DNA (column and magnetic beads DNA recovery system) and ELISA detection methods were used to detect soy in heated and non-heated food matrices. The limit of detection for column DNA recovery method in soybean, SPI and soymilk can be as low as 20 ppm, while magnetic beads DNA method was matrix dependent. The magnetic beads methods demonstrated a lower detection for soybean sample (1.33 ppm) but higher for soymilk (133.3 ppm). The soy percent recovery for non-heated food matrices was higher in ELISA methods and lower in magnetic beads DNA method. For heated food matrices, percent recovery for both DNA methods was higher than ELISA method. Overall, heat treatment can significantly reduce the ability of the ELISA method to detect soy in all food matrices. However, for DNA methods (column and magnetic beads), water and ranch matrices were the only two that were significantly affected by thermal processing. In terms of food matrices, water matrix (heated and non-heated) has the highest percent recovery of soy for all detection methods. However, percent recovery of soy in flour matrix (non-heated) was the lowest using both DNA methods. / Graduation date: 2013

Pseudomonas fluorescens EPS62e es va seleccionar com a agent de biocontrol del foc bacterià per la seva eficàcia en el control de Erwinia amylovora. En aquest treball es van desenvolupar mètodes de traçabilitat que van permetre la seva detecció específica i quantificació. Mitjançant les tècniques RAPD i U-PCR es van obtenir fragments d'amplificació diferencial per EPS62e que es van seqüenciar i caracteritzar com marcadors SCAR per dissenyar una PCR en temps real. La PCR a temps real es va utilitzar simultàniament amb mètodes microbiològics per estudiar l'adaptabilitat epifítica de EPS62e en pomera i perera. L'ús combinat de mètodes microbiològics i moleculars va permetre la identificació de tres estats fisiològics de EPS62e: la colonització activa, l'entrada en un estat de viable però no cultivable, i la mort cel·lular. Aquest treball mostra que EPS62e està ben adaptada a la colonització de flors a camp, encoratjant la seva utilització dins d'una estratègia de control biològic contra el foc bacterià. / Pseudomonas fluorescens EPS62e was selected as a reliable biological control agent of fire blight for its high efficacy controlling Erwinia amylovora infections. In the present work, monitoring methods which allowed EPS62e specific detection and quantification were developed. RAPD and U-PCR fingerprints were used to obtain differential amplified fragments from EPS62e that were sequence characterized as SCAR markers. A real-time PCR was developed on the basis of the strain-specific SCAR markers, and was used simultaneously with microbiological methods to study the environmental fate of EPS62e in apple and pear orchards. The combined use of both microbiological and molecular methods permitted the identification of three physiological states for EPS62e, which consisted of active colonization, survival and entry into a viable but nonculturable state, and cell death. The present work shows that EPS62e is well adapted for blossom colonisation in the field, and encourages its utilisation in a fire blight.

Vaginitis is a vaginal infection which affects many women all over the world. The disorder is characterized by an infection of the vaginal area which can cause problems like abnormal vaginal discharge, itching and redness. The two most common causes of vaginitis are bacterial vaginosis and Candida vaginitis. The prevalence of bacterial vaginosis in Sweden is around 10-20 % and approximately 75 % of all women will once in their lifetime suffer from vaginal yeast infection. The clinical symptoms of vaginal infections are not specific and the diagnosis methods of bacterial vaginosis and Candida vaginitis are subjective and depended on the acuity of the clinician. Due to the lack of standardized and objective diagnostic tools, misdiagnosis and consequently incorrect treatment may occur. As vaginal infections and symptoms impact greatly of women´s quality of life and vaginitis have been associated with serious public health consequences, it is essential to diagnose and treat the conditions correctly. Hence, there is a great need of better methods of diagnosing these conditions. The aim of this master thesis was to develop quantitative species-specific real-time PCR assays to use in diagnosing the two most common causes of vaginitis i.e. bacterial vaginosis and Candida vaginitis. Potential markers for bacterial vaginosis (Atopobium vaginae, BVAB2, Gardnerella vaginalis, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, Megasphaera type 1, Megasphaera type 2, Mobiluncus curtisii, Mobiluncus mulieris and Leptotrichia/Sneathia species) and Candida vaginitis (Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis) were chosen. Primers and probes were designed and tested on reference strains and vaginal samples. Single- and multiplex PCR reactions were successfully optimized with the designed oligonucleotides. Furthermore, standard curves with excellent linearity were created and covered more than five orders of magnitude. These developed quantitative species-specific real-time PCR assays will, in a prospective medical validation, quantify 300 vaginal samples from women visiting the RFSU Clinic in Stockholm.

This study was carried out to analyze tomato samples and tomato seeds, purchased from different food markets of Turkey randomly, for the presence of genetic modification by using PCR method as it allows more specific detection. The DNAs of collected samples were isolated according to CTAB DNA extraction protocol and also with extraction kits. Screening tests of tomatoes were done by targeting 35S promoter, NOS terminator and NptII kanamycin resistance gene with eight different primer sets. Real time PCR is used to confirm 35S and NOS positives results obtained from conventional PCR. In this study, it was observed that 14 out of 35 seed samples, and 14 out of 40 fresh tomato samples which were screened had at least one transgenic element of 35S promoter, NOS terminator and NPTII kanamycin resistance gene indicating the possible presence of genetic modifications. After screening, gene specific studies were carried out for PG, sam-k indicating F type ripening delayed tomato and the 35 1 N lines respectively and cry1Ac genes inserted in 5345-1 insect resistant tomato line. PG and sam-k specific primers were not amplified in any of the samples investigated whereas 18 out of 75 samples were cry1Ac positive and 1 out of 75 samples was sam-k positive. Positives were confirmed by sequence analysis. Additionally, construct specific primers specific to 5345-1 and 35 1 N lines were designed. PCR amplicons indicate the existence of the construct sequence. In order to verify the results, PCR products were sent to sequence analysis

The effects of diabetes on cytochrome P450 dependent drug metabolizing enzymes have not to be clarified yet. The most widely used animals in these studies have been rats, and information regarding the effects of diabetes on cytochrome P450 dependent procarcinogen/carcinogen metabolism in rabbits is limited. In the present study, we investigated, for the first time, the influence of benzene on liver, kidney and lung microsomal cytochrome P450 dependent drug metabolizing enzyme activities, protein and mRNA levels in diabetic and non-diabetic rabbits. Male New Zealand rabbits were made diabetic by a single dose of alloxan treatment in this study. AST, ALT and LDH enzyme activities in the blood serum and lipid peroxidation in liver microsomes were found to increase in diabetic, benzene treated and benzene treated diabetic rabbits. Besides these, CYP2E1 dependent NDMA N-demethylase and p-nitrophenol hydroxylase activities and CYP2E1 protein level were found to increase in liver and kidney of diabetic and benzene-treated rabbits. The combined effects of benzene and diabetes on these activities and protein level were found to be additive. Although diabetes caused induction of pulmonary CYP2E1 protein level and associated enzyme activities, benzene treatment of rabbits resulted in no change in enzyme activities and protein level in lung. The level of mRNA was investigated by Real-Time PCR. Accordingly, hepatic CYP2E1 mRNA level was increased 6.71-, 10.53- and 12.93-fold in diabetic, benzene treated and benzene treated diabetic rabbits with respect to the control animals. Similarly, renal CYP2E1 mRNA level was found in increase in these rabbits. In addition to CYP2E1, CYP3A6 associated enzyme activity, erythromycin N-demethylase, CYP3A6 protein and mRNA level were found to increase in diabetic rabbit liver and lung. Unlike diabetes, benzene treatment caused suppression of CYP3A6 protein and inhibition of associated enzyme activity in liver. There was no significant change in the erythromycin N-demethylase activity and CYP3A6 level of liver and lung as a result of benzene treatment of diabetic rabbits. Moreover, diabetes induced CYP1A2 protein and mRNA level and CYP1A associated enzyme activities in the rabbit liver. On the other hand, benzene caused statistically insignificant decreases in CYP1A dependent enzyme activities and CYP1A2 protein level in liver. CYP1A associated enzyme activities, CYP1A2 protein and mRNA levels were not changed in the liver of benzene treated diabetics. The results of the present work indicate that both diabetes and benzene stimulate metabolic activation toxic chemicals metabolized by CYP2E1 such as NDMA and benzene by inducing CYP2E1 which results in the formation of increased amounts of reactive metabolites. Application of benzene to diabetic rabbits further elevates expression and activities of the CYP2E1. As a result of additive induction of the CYP2E1 in benzene treated diabetics, further increase the risk of hepatotoxicity produced by toxins may be observed when compared to the separate treatments. This may in turn further potentiate the risk of organ toxicity and mutagenesis in liver and kidney of these subjects. As in the case of CYP2E1, the risk of carcinogenesis due to induction of CYP1A may be increased in diabetic subjects. Moreover, in diabetic and benzene exposed subjects, alteration of drug clearance and clinical drug toxicity may be observed due to induction or suppression of CYP3A.

Although traditional microbiological methods are accepted standard for Salmonella detection, they are labor intensive and time consuming. Therefore, for food industry and public health, finding sensitive and rapid methods is required. As a rapid and reliable tool, Real-Time PCR is one of the most common methods in molecular detection and research area. The aim of the current study is to develop rapid, sensitive and quantitative Salmonella detection method using Real-Time PCR technique based on inexpensive, easy to produce, convenient and standardized plasmid based positive control for the first time. To achieve this, two plasmids were constructed as reference molecules by cloning two most commonly used Salmonella specific target regions &lsquo / invA and ttrRSBC&rsquo / into them. Standard curves were constructed for the plasmids and reproducibility, PCR efficiency, amplification efficiency values were calculated. To illustrate the applicability of the developed method, enriched (as used commonly for Salmonella detection with Real-Time PCR) 105 to 100 CFU/ml level (estimated by standard plate counts before enrichment) S. Typhimurium ATCC 14028 cultures were tried to detect and quantify, also compared with traditional culture method. In addition, detection limits of the developed technique were determined by serial dilution of DNA extracted from 105 CFU/ml level. The results revealed much faster detection ability of the developed plasmid based Salmonella detection method (in comparison to traditional culture method, ISO 6579:2004) allowing quantitative evaluation with perfect reproducibility, sensitivity (except for lower concentrations for invA target), detection limit, PCR efficiency, amplification efficiency for both invA and ttrRSBC targets. The detection and quantification ability of the method developed by using S. Typhimurium ATCC 14028 cultures were tested also with 15 Salmonella species using milk as a representative food. The results also revealed much faster (in comparison to traditional culture method, ISO 6579:2004) quantitative detection ability of the developed method. Thus, the developed method has great potential to be used in food industry for rapid and quantitative Salmonella detection.

Autoinducer 2 (AI-2) is a quorum sensing signal employed by bacteria to coordinate their response to environmental stresses. The objective of this study was to determine the relationship between presence of AI-2 molecules, exposure to sub-therapeutic tetracycline, the expression of genes associated with the conjugal transfer of antibiotic resistance plasmids, and the conjugal transfer of these plasmids in Escherichia coli. The studies showed that AI-2 activity increased in Tets E. coli in the presence of tetracycline (2 µg/mL) under both batch and continuous culture conditions. The presence of AI-2 molecules induced tetracycline tolerance development in Tets E. coli. The studies showed that the survival rates of Tets E. coli exposed to AI-2 molecules were significantly higher compared to the cells not exposed to AI-2 molecules or cells that were exposed to only LB (Lauria-Bertani) broth. Molecular analyses using real-time PCR indicate that the expression of at least one conjugation-associated gene (trbC) is increased 9-fold in cells exposed to AI-2 molecules in the presence of sub-therapeutic tetracycline compared to its negative controls. The transconjugation frequency of the plasmid RP4 carrying the tet(A) gene increased between 10-100 fold in the presence of AI-2 molecules. In companion studies, AI-2-like activity was detected in fish, tomatoes, cantaloupes, carrots and milk samples. Interestingly, ground beef and poultry meat contained substances that appear to inhibit AI-2 activity. Collectively, these results highlight the potential importance of bacterial quorum sensing signals such as AI-2 in the response of bacterial cells to environmental stimuli and the possible role of quorum sensing signals in the quality and safety of foods.

ABSTRACT A defect implantation process is the major reason for unexplained infertility. Estrogen andprogesterone are steroid hormones preparing the endometrium for implantation. They mediatetheir effect through their receptors: estrogen receptor alpha and beta and progesteronereceptor A and B, respectively. Leukemia inhibitory factor (LIF), which is also important forimplantation, mediates its effect through LIF receptor and the coreceptor, gp130, and is downregulated by suppressors of cytokine signaling 1. The aim of the study was to compare thelevels of the steroid hormone receptors and LIF related factors in the endometrium of twogroups of women with the diagnosis unexplained infertility: one that became pregnant afterassisted reproduction and one that did not become pregnant. Before treatment of thesewomen, endometrial mRNA was collected during the window of implantation in themenstrual cycle. The levels of specific mRNAs were measured with real-time PCR. Womenwho had become pregnant had a significantly higher level of steroid hormone receptors. Thus,these proteins seem to be important for a pregnancy and may be suitable as receptivitymarkers.