Mould is a common contamination in cereals. The growth of mould can stimulate mycotoxins production andsome of which at critical concentrations cause health problems in humans and animals. Fusarium is one of thefungus species that has been found in crops and can cause major problems for farmers such as reduced harvestand economic losses. A group of Fusarium species, Fusarium avenaceum, Fusarium poae and Fusariumtricinctum express a mycotoxin, enniatin. The limited information available today about enniatin-forming fungiis that they grow out on fields of wheat in colder climates. This project aims at developing methods for detection,quantification and identification of known and unknown fungi present in Swedish cereals during 2009-2011. Theproject was carried out using two previously published methods, TMAV and MGB, which both use TaqManprobes with realtime-PCR detection. The methods were evaluated for robustness, efficiency, accuracy, inclusionand exclusion. The results showed that both methods, TMAV and MGB, could be used to detect Fusariumavenaceum. The results for the TMAV method were that it could be used with custom annealing temperatureand chemical concentrations for the best detection. The MGB method can be used to detect Fusarium avenaceumwith Fusarium tricinctum in the same analysis. Both methods can be included in future mapping projects when itis of interest to quantify enniatin producing moulds.

The aim of this study was to access the efficacy of an enhanced in situ bioremediation technology at a 1,2-dichloroethane (1,2-DCA) polluted site in southern Taiwan. A water-soluble substrate was injected into the groundwater to provide carbon sources for microbial growth. After substrate injection, increased total organic carbon (TOC) concentrations and microbial populations including Dehalococcoides spp. and Desulfitobacterium spp. were observed in the groundwater. Microbial diversity was analyzed using denaturing gradient gel electrophoresis (DGGE) and 16S rDNA sequencing to identify the bacterial strains. The results showed that after 4.5 months of substrate injection, the reduction-oxidation potential (ORP) changed from aerobic to anaerobic conditions. The less oxygen-tolerable 1,2-DCA degrading bacteria Dehalococcoides spp. started to accumulate in groundwater. However, the more oxygen-tolerable Desulfitobacterium spp. didn¡¦t show a prominent change, although the ORP was suitable for Desulfitobacterium spp. to carry out reductive dechlorination. The DGGE results indicate that with the injected carbon sources and mineral nutrients, both the groundwater microbial diversity and the amount of dominant bacteria were increased. The 16S rDNA sequencing demonstrated that the amount and diversity of 1,2-DCA degradation-related bacteria also increased with the injection of substrate. Six groups of 1,2-DCA degradation related reactions were found: dechlorination, chlorinated-compound degradation, denitrification, iron-reduction, sulfate-reduction and methane-utilizing. Four species that can directly degrade 1,2-DCA were found: Dehalobacter sp., Dehalococcoides sp., Nitrosospira sp. and Pseudomonas sp. Moreover, 11 methane-utilizing bacterial species were also discovered. The presence of these methane-utilizing bacteria not only might assist the process of denitrification and sulfate-reduction, but also could diminish the emission of the greenhouse gas. The results of this study confirmed that the addition of substrates could affect the groundwater oxidation-reduction state and enhance the bioremediation at the 1,2-DCA-contaminated site. Thus, enhanced in situ bioremediation is a feasible technology for site remediation.

An emerging trend towards non-laboratory based biological and microbiological marker analysis is occurring in multiple sectors of science and industry. In the medical sector, these trends have demonstrated that conducting sample analyses away from centralised laboratories not only makes analyses quicker and more convenient (e.g. a home pregnancy test), but can offer services that are otherwise impractical (e.g. mobile laboratories to diagnose disease in the developing world). In the environmental sector, similar benefits, plus the ability to develop and test hypotheses, protocols and sampling strategies within a field campaign, are possible with in-field analyses. Icy environments in particular would benefit from in situ or in-field life detection as they are typically remote, and hence impart high logistical costs for repeated field campaigns and associated sample return with the implication that the efficiency of scientific return is poor. Unfortunately, most equipment and protocols developed for microbiological analyses in other sectors of science and industry are unsuitable for direct application to in-field use in icy environments because of poor compatibility with icy environment sample matrices and frequently inappropriate microbiological targets. Hence within this work, two hypotheses were tested: that (i) microbiological detection infield in icy environments is possible and through this (ii) unique and more efficient scientific studies can be conducted. Cont/d.

<p>Directive EC 2003/89/EC of the European Parliament and of the Council states that certain</p><p>ingredients and products derived there of known to cause allergen reactions must always be</p><p>declared. Furthermore labelling is mandatory irrespective of the amount included. The National</p><p>Food Administration therefore needs methods for monitoring the presence of allergens in food.</p><p>Methods already exist for most of the allergens on the EU-list, but an operational method for</p><p>celery (Apium graveolens) is missing.</p><p>A specific DNA-method was developed, based on TaqMan Real-Time PCR with the celery</p><p>mannitol dehydrogenase gene as target sequence. The analysis was started with homogenisation</p><p>of the sample followed by extraction of DNA. The Real-Time PCR method was shown to be</p><p>specific for celery, producing a 113 bp fragment with two celery varieties and negative results</p><p>with other closely selected species commonly present together with celery in food products (12</p><p>samples). The detection limit was 2-20 pg DNA, which corresponds to 1-7 haploid genome</p><p>copies. When evaluated with model samples of celery in meat, a detection limit of less than</p><p>0,01 % was determined. When used to analyse food products from the market, six out of seven</p><p>products declared to contain celery were correctly identified as positive.</p>

Directive EC 2003/89/EC of the European Parliament and of the Council states that certain ingredients and products derived there of known to cause allergen reactions must always be declared. Furthermore labelling is mandatory irrespective of the amount included. The National Food Administration therefore needs methods for monitoring the presence of allergens in food. Methods already exist for most of the allergens on the EU-list, but an operational method for celery (Apium graveolens) is missing. A specific DNA-method was developed, based on TaqMan Real-Time PCR with the celery mannitol dehydrogenase gene as target sequence. The analysis was started with homogenisation of the sample followed by extraction of DNA. The Real-Time PCR method was shown to be specific for celery, producing a 113 bp fragment with two celery varieties and negative results with other closely selected species commonly present together with celery in food products (12 samples). The detection limit was 2-20 pg DNA, which corresponds to 1-7 haploid genome copies. When evaluated with model samples of celery in meat, a detection limit of less than 0,01 % was determined. When used to analyse food products from the market, six out of seven products declared to contain celery were correctly identified as positive.

The climate in the north is changing over time, which affects the nature in many ways. For instance, some microorganisms that cause infections might become more common. This might have negative consequences for reindeer husbandry. In Sweden, this is an industry that is relatively large. However, even though the reindeer is common in the north the knowledge about its diseases is limited.In this study the prevalence of microorganisms that may cause infection in reindeer was investigated. Comparisons between different sami villages and previous studies were performed to detect differences that could occur due to climate changes. The diseases and microorganisms that were analyzed with PCR were malignant catarrhal fever, herpes infections, Chlamydia sp. and bovine viral diarrhea (BVD). The cause of eye problems in reindeer was also investigated. BVD and bovine leukemia virus where analyzed with ELISA. Next generation sequencing where used for broader screening of samples for microorganisms that might be of interest of future analysis in more detailed follow-up studies.Since not enough samples were available at the time of this study findings could not be linked to changing climate. In the reindeer with eye infection Chlamydia sp., Moraxella sp. and Neisseria sp. can probably be involved causing disease. This should be further investigated to be able to determine whether it is true or not by analyzing samples from individuals without changes in the eyes. The prevalence of reindeers with antibodies against BVD has increased in Sweden since 2012. There will be further studies in this field with reindeers from other northern countries.

Resumo: Os prejuízos causados à pecuária brasileira pelo carrapato (Rhipicephalus (B.) microplus) são significativos e, uma das estratégias utilizadas para aumentar a produtividade dos rebanhos nas regiões tropicais tem sido a utilização de raças mais resistentes a esse ectoparasita. O objetivo desse projeto foi aplicar a tecnologia de microarray de DNA para a prospecção de genes relacionados com os mecanismos de resistência/tolerância ao carrapato, mediante a análise da expressão gênica diferencial em linfonodos de bovinos suscetíveis (Aberdeen Angus) e resistentes (Nelore) infestados artificialmente com este parasita. Os bezerros foram mantidos livres de carrapato desde o nascimento e foram infestados artificialmente com larvas de carrapato aos quatro meses de idade. As biópsias de linfonodo foram realizadas antes e após a infestação e mantidas a -80°C até o seu processamento. Essas amo stras foram submetidas a extração de RNA, síntese de cDNA e marcação . Depois elas foram submetidas a hibridização pela técnica de microarray. Foram identificados 341 genes diferencialmente expressos nos bovinos da Raça Angus e 254 para bovinos da Raça Nelore, os quais foram agrupados em categorias funcionais. Foram identificadas diferenças no padrão de expressão de genes de resposta imune entre as raças, tais como: Moléculas CD, Imunoglobulinas, Fator de Necrose tumoral, Integrinas e Interferon-γ. Para validação dos resultados de expressão diferencial foi empregada a técnica de PCR em tempo real, onde se verificou a expressão dos genes das citocinas IL-5 e IL12p40 nas duas Raças estudadas. / Abstract: Significant losses are brought by ticks (Rhipicephalus (B.) microplus) to Brazilian beef farming. One of the strategies to increase yield in tropical regions is the use of resistant breeds. This study was undertaken to apply the DNA microarray technology for the prospection of genes related to tick resistance/tolerance mechanisms, through differential gene expression analysis in lymphonodes of susceptible (Aberdeen Angus) and resistant (Nelore) breeds artificially infested with the parasite. Calves were kept tick-free since birth and infested with tick larvae when they reached four months of age. Lymphonode biopsies were performed before and after infestation and kept at -80°C until analyses. Samples were subjected to RNA extraction, cDNA synthesis and labelling. Hybridization was then performed through the microarray technique. In Angus, 341 differentially expressed genes were found against 254 in Nelore, which were grouped in functional categories. Different expression patterns were identified immune response genes between breeds, such as: CD molecules, Immunoglobulines, Tumor Necrosis Factor, Integrins and Interferon-γ. Real-time PCR was used to validate results, which showed the expression of cytokines IL-5 and IL12p40 in both breeds. / Orientador: Luiz Roberto Furlan / Coorientador: Márcia Cristina de Sena Oliveira / Banca: Maria Inês Tiraboschi Ferro / Banca: Maribel Elizabeth Funes Huacca / Mestre

Typning av hla-b*27:En jämförelsestudie mellan två analysmetoder för att påvisa HLA-B*27 molekylen i ankyloserande spondylitCarolina BermudezBermudez, C. Typning av HLA-B*27. En jämförelsestudie mellan två analysmetoder för att påvisa HLA-B*27 molekylen i Ankyloserande spondylit. Examensarbete i Biomedicinsk vetenskap, 15 högskolepoäng. Malmö universitet: Fakulteten för hälsa och samhälle, institutionen för Biomedicinsk vetenskap, 2018.Human leukocyt antigen (HLA) är vävnadsantigener, belägna på våra vita blodkroppar. HLA-B*27 allelen är starkt kopplat till Ankyloserande spondylit (AS). Det är en kronisk inflammatorisk ledsjukdom, som främst attackerar ryggraden, bäckenet och bröstkorgen. Det finns idag ingen enskild laborativ metod som med full säkerhet kan fastställa diagnos av denna sjukdom, innan de kliniska symtomen uppträder. Typning av HLA-B*27 ger endast information om närvaro eller frånvaro av antigenet, vid utredning av AS. Vidare är HLA-B*27 en polymorf och de olika alleltyperna varierar kraftigt, bland skilda etniska grupper samt mellan geografiska områden. Genetiska- och miljöfaktorer påverkar också. Sjukdomsutveckling i samband med närvaro av HLA-B*27 allelen, varierar därför från individ till individ. Därmed fungerar metoden endast som ett komplement-verktyg, för att ytterligare bekräfta diagnos. Syftet med denna studie var att med realtids-polymerase chain reaction (PCR), utföra typning av HLA-B*27 med Linkseq kit samt jämföra analysresultaten med uthämtade resultat från intern sjukhusdatabas, där typning av HLA-B*27 hade utförts med PCR-SSP (sekvens-specifika primers). Samtliga resultat stämde överens till 100%, vilket indikerar att metoden fungerar bra. Det finns studier som visat att HLA-B*27 molekylens fria tunga kedjor (HLA-B*272) har en starkare benägenhet än andra HLA-molekyler att binda in till killer immunoglobine-like receptorer (KIRs). Inbindning till KIRs med efterföljande ökad stimulering av interleukiner (IL) främst IL-17 och IL-23 bidrar till sjukdomsutvecklingen av AS. Dock finns ingen HLA-B*272 specifik antikropp som kan bevisa detta och det behövs därför ytterligare undersökning för att hitta en sådan. Därefter skulle en ny laborativ metod kunna utvecklas för att fastställa diagnos av AS i ett tidigt skede, innan de kliniska symtomen uppvisas. Nyckelord: Allelvarianter, Ankyloserande spondylit, HLA-B*27, KIR, PCR-SSP, Realtids-PCR. / typing of hla-b*27:a comparison study between two analysing methods for the detection of the HLA-B*27 molecule in ankylosing spondylitisCarolina BermudezBermudez, C. Typing of HLA-B*27. A comparison study between two analysing methods for the detection of the HLA-B*27 molecule in Ankylosing spondylitis. Degree project in Biomedical Laboratory Science, 15 credit points. Malmö University: Faculty of Health and Society, Department of Biomedical science, 2018.Human leukocyte antigen (HLA) are tissue antigens located on our white blood cells. The HLA-B*27 allele is strongly related to Ankylosing spondylitis (AS). It is a chronical inflammatory rheumatic disease that primarily affects the spine, the pelvis and the chest. At present, there is no single laboratory method that with all certainty may determine diagnosis of this disease, before the clinical symptoms appear. Typing of HLA-B*27 only gives information about the presence or absence of the antigen, upon the investigation of AS. Furthermore, HLA-B*27 is a polymorph and the different types of alleles, strongly vary among different ethnic groups and also between geographic regions. Genetic- and environmental factors also affect. Development of disease in conjunction with the presence of the HLA-B*27 allele, therefore varies from one individual to another. So, the method only functions as a complementary tool, to further confirm diagnosis. The aim of this study was to perform HLA-B*27 typing with realtime-polymerase chain reaction (PCR) using Linkseq kit and compare the analysed results with those results that were retrieved from the internal database of the hospital, where typing of HLA-B*27 had been performed with PCR-SSP (sequence specific primers). All results agreed with 100%, which indicates that the method functions well. There are studies that show that the heavy chains (HLA-B*272) of the HLA-B*27 molecule have a stronger affinity than other HLA-molecules of binding in to killer immunoglobulin-like receptors (KIRs). Increased stimulation of interleukins (IL) primarily IL17 and IL23, following binding to KIRs, contributes to the pathogenesis of ankylosing spondylitis. However, there is no HLA-B*272 specific antibody that may prove this and therefore more investigation is needed, in order to find one. A new laboratory method could then be developed to determine diagnosis of AS at an early stage, before the clinical symptoms emerge. Keyword: Allelvariants, Ankylosing spondylitis, HLA-B*27, KIR, PCR-SSP, Realtime-PCR.

Autoimmunitet är ett tillstånd där kroppens immunsystem felaktigt attackerar och skadar sina egna vävnader och celler. HLA-B27 är en genvariation som kan kopplas till autoimmun sjukdom som ankyloserande spondylit med en prevalens på 2-4% i världens befolkning. Denna studie syftade till att utvärdera och jämföra två kit för HLA-B27 alleler mot den nuvarande metoden på Länssjukhuset Ryhov i Region Jönköpings län. De metodprinciper som användes var realtids-PCR samt LAMP.  Totalt analyserades 37 avidentifierade blodprover med vardera av kiten samt med nuvarande metod. Resultatet visade en överensstämmelse med avseende på förväntade positiva och negativa resultat för HLA-B27 för de två kiten jämfört med nuvarande metoden. De tre metoderna/kiten detekterar de vanligaste HLA-B27 allelerna. Utifrån studiens resultat visade sig båda kiten vara effektiva, lättanvända samt ha stabila reagenser. Dessutom uppnådde båda kiten de IVD-krav som ställs inom EU. / Autoimmunity is a condition where the body's immune system mistakenly attacks and damages its own tissues and cells. HLA-B27 is a genetic variation that can be linked to autoimmune diseases such as ankylosing spondylitis, with a prevalence of 2-4% in the world’s population. This study aimed to evaluate and compare two kits for HLA-B27 alleles against the current method at Ryhov County Hospital in Region Jönköping County. The methodological principles used were real-time PCR and LAMP. A total of 37 anonymized blood samples were analyzed using each of the kits and the routine method. The results showed concordance with the expected positive and negative results for HLA-B27 between the two kits compared to the current method. The three methods/kits detect the most common HLA-B27 alleles.  Based on the study’s results, both kits proved to be effective, user friendly, and have stable reagents. Additionally, both kits met the IVD requirements set within the EU.