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No. of bitstreams: 2 Dissertação - Anniely Carvalho Rebouças Oliveira - 2016.pdf: 1878998 bytes, checksum: 1afecfd5948d2abb2a5374cbeb9ff005 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The acute respiratory infections (ARIs) are an important cause of morbidity and mortality worldwide. Viruses have an important role in the etiology of the respiratory infections. Children less than five years of age have about four to six ARIs per year and this is a common cause of hospitalization, mainly in developing countries. In Brazil, particularly in the West-Central region, studies that evaluated the circulation of respiratory viruses in the pediatric population are scarce. Thus, this study aimed to investigate the occurrence of respiratory viruses in the pediatric population, from Goiânia-Goiás. Between May/2014 and May/2015, samples of nasal swabs were collected from children from zero to six years of age presenting or not respiratory symptoms, attended at the Materno Infantil hospital in Goiânia. For the molecular screening, 16 viruses were investiged using three Multiplex Nested-PCR protocols. It two hundred and fifty one samples were collected, of wich 90 (35,9%) were positive for at least one respiratory agent, being rhinovirus (31%), respiratory syncytial virus (27,4%) and parainfluenza virus (13,3%) the most prevalent. Children under two years of age had greater overall positive, although not statistically significant. Similar detection rate was observed among the groups symptomatic (37%) and asymptomatic (34,5%). For the symptomatic group, children whose samples were collected during the dry period (p<0,05) and those with up to two years of age were most affected (p<0,05). In the asymptomatic group no statistical difference was observed. It was observed rate of co-detection of 6,4% (16/251), mostly in samples of symptomatic patients (13/16) (p<0,05). The evaluation of the temporal profile showed a higher detection rate during the dry season, less rainfall, relative humidity and cooler temperatures. The results reinforce the importance of respiratory viruses in children and contribute to further understanding of the epidemiological and temporal factors associated with different pathogens in our region. Thus, they open the way to new studies in the state of Goiás, also providing information to assist in the construction of control measures and more effective prevention of these infections. / As infecções do trato respiratório (ITRs) representam importante causa de morbimortalidade em todo o mundo, sendo os vírus importantes agentes etiológicos. Estima-se que grande parte das crianças menores de cinco anos de idade tenha a cada ano de quatro a seis ITRs, sendo esta causa comum de hospitalização, especialmente em países em desenvolvimento. No Brasil, em particular na Região Centro-Oeste, estudos que avaliem a circulação de vírus respiratórios em população pediátrica são escassos. Neste contexto, o presente estudo teve como objetivo investigar a ocorrência de diferentes vírus respiratórios em população pediátrica de Goiânia-Goiás, apresentando ou não quadro sintomático respiratório. Durante o período de maio/2014 a maio/2015, foram coletadas amostras de swab respiratório de crianças entre zero e seis anos de idade, apresentando quadro de infecção respiratória ou assintomáticas para a mesma, atendidas no Hospital Materno Infantil, em Goiânia. As amostras foram submetidas à triagem molecular para 16 vírus respiratórios por meio de três protocolos de multiplex nested-PCR. No total, 251 amostras foram coletadas, das quais 90 (35,9%) apresentaram positividade para pelo menos um agente respiratório, sendo rinovírus (31%), vírus sincicial respiratório (27,4%) e parainfluenza (13,3%) os mais frequentes. Crianças com até dois anos de idade apresentaram maior positividade global, contudo sem diferença estatística. Índice de detecção semelhante foi observado entre os grupos sintomático (37%) e assintomático (34,5%). Para as crianças do grupo sintomático, foram mais afetadas aquelas cujas amostras foram coletadas durante período de seca (p<0,05) e aquelas com até dois anos de idade (p<0,05). No grupo assintomático, nenhuma diferença estatística foi observada. Foi observado índice de co-detecção de 6,4% (16/251), sendo a maioria de indivíduos sintomáticos (13/16) (p<0,05). A avaliação do perfil temporal demonstrou maior índice de detecção nos meses de seca, com menor precipitação pluviométrica, umidade relativa do ar e temperaturas mais frias. Os resultados obtidos no presente estudo reforçam a importância dos vírus respiratórios na população pediátrica, auxiliando na compreensão dos fatores epidemiológicos e temporais associados aos diferentes patógenos em nossa região. Desta forma, abrem caminhos para novos estudos no estado de Goiás, fornecendo ainda informações que auxiliem na construção de medidas de controle e prevenção mais eficazes contra estas infecções.

<p>Initially, a pilot study was carried out in order to reconstruct the major paternal and maternal lineages of the Muslim population living in the Cape metropolitan area. The Study has shown the ability of molecular genetic tools to give insight into the origins and history of local communities. The study was also used as a point of reference for the Strand Muslim Community project. Genetic variations of the Y-chromosome and mitochondrial DNA for the pilot study were analyzed using the RFLP technique. The SNaPshot mini-sequencing technique was used to genotype single nucleotide polymorphisms (SNP) on the Y-chromosome and mitochondrial DNA in 115 males from the Strand Muslim community.</p>

This dissertation includes molecular and phytochemical investigations of the harmful, bloom-forming alga, Prymnesium parvum, including analysis of known polyketide metabolites as a function of salinity and growth. Initially, the development of molecular and phytochemical tools was necessary for the detection and quantification of P. parvum and its associated toxins. Suites of oligonucleotides and molecular beacons were designed for conventional and quantitative multiplex PCR to amplify four species- and gene-specific products simultaneously that were used for the detection and quantitation of P. parvum. This built-in redundancy provided increased confidence in reactions with the positive confirmation of four discrete products. Techniques were also developed for the chemical enrichment of toxins produced by P. parvum. Until now, isolation of “prymnesins” has never been reproduced. Polyketide prymnesins possess unique spectral properties that were used to generate an LC-MS fingerprint that comprised 13 ion species. Preliminary investigations using chemifluorimetric methods were also capable of detecting prymnesins in the pico- and nano-molar range. Environmental samples were tested as an independent assessment of these methods. Lastly, the roles of polyketide prymnesins were analyzed with respect to total hemolytic activity (HA) as a function of culture age and salinity. Variation in HA of supernatants was statistically significant relative to both variables (p << 0.05). Salinity was inversely related to HA wherein cultures growing in 5-25 psu were 150-200% more hemolytic. Total HA was inversely related to culture age during the first three weeks, but positively related to it during the next three weeks. Interestingly, no hemolysis was detected in fractions containing prymnesins from culture supernatants and the majority of hemolysins remained in the aqueous phase. Prymnesins extracted from cells varied significantly over the 6-week observation period (p << 0.05); HA was positively correlated during the first half and inversely related during the last half of the study. Salinity was directly related to HA from cell extracts, but these effects were not significantly different until the last three weeks. These investigations suggest that polyketide prymnesins are present at much lower quantities than previously believed, and they may not be the key compounds associated with hemolysis due to P. parvum. / text

<p>Initially, a pilot study was carried out in order to reconstruct the major paternal and maternal lineages of the Muslim population living in the Cape metropolitan area. The Study has shown the ability of molecular genetic tools to give insight into the origins and history of local communities. The study was also used as a point of reference for the Strand Muslim Community project. Genetic variations of the Y-chromosome and mitochondrial DNA for the pilot study were analyzed using the RFLP technique. The SNaPshot mini-sequencing technique was used to genotype single nucleotide polymorphisms (SNP) on the Y-chromosome and mitochondrial DNA in 115 males from the Strand Muslim community.</p>

Respiratorische Krankheitsbilder, an denen mehr als ein Pathogen ursächlich beteiligt ist, gewinnen in der Schweinepopulation zunehmend an Bedeutung. Diagnostische Methoden zum simultanen Nachweis mehrerer Erreger sind Bestandteil einer schnellen und effizienten Therapie und tragen zum ökonomischen Bestandsmanagement bei. Im Rahmen dieser Arbeit sollten Methoden für den Multiplex-Nachweis von Antikörpern und Nukleinsäuren viraler Erreger von respiratorischen Krankheitsgeschehen des Schweins entwickelt werden. Die Methode für den Multiplex-Nachweis von Antikörpern sollte auf Basis der xMAP® Flüssigchip-Technologie (Luminex Corporation, Austin, T, USA) an der LiquiChip®- Workstation (Qiagen, Hilden, D) etabliert werden. Da es sich um eine für den Antikörpernachweis im veterinärmedizinischen Bereich bislang nicht genutzte Methode handelte, erfolgte die Prüfung der Machbarkeit zunächst im Einfach-Format am Beispiel des Porzinen Circovirus Typ 2. Im Laufe der Arbeit wurde deutlich, daß die Kopplung des PCV2 ORF2-Proteins als Capture-Molekül sowie die Erstellung der Versuchsansätze mit akzeptablem Aufwand ohne Spezialtechniken durchführbar war. Aufgrund der Anordnung der Proben auf Platten im 96-well-Format und der vollautomatischen Messung war ein hoher Probendurchsatz möglich. Nach der Einführung von Waschschritten in die Versuchsansätze konnten hohe Fluoreszenzsignale erzeugt werden. Im Laufe der Optimierungsversuche wurde allerdings die fehlende Korrelation dieser Fluoreszenzsignale mit den Ergebnissen der Referenzmethode deutlich. Aufgrund der unbekannten Testeigenschaften sowie fehlender Kontrollmöglichkeiten wurden diese nicht sogleich als unspezifische bzw. falsch positive Signale erkannt. Erst durch die Testung von positiven und negativen Feldseren an verschiedensten Bead-Arten wurde ersichtlich, daß die Fluoreszenzen nicht ausschließlich durch die spezifische Bindung der PCV2-Antikörper an das Capture-Protein entstanden. Im Ausschlussverfahren konnte die Ursache eingegrenzt werden. Bestandteile aus dem Schweineserum führten vermutlich durch unspezifische Bindungen an die LiquiChip®-Beads zu einem Fluoreszenzereignis. Durch Vorinkubation der Beads in Pferdeserum und der Feldseren mit einem Block-Puffer wurde versucht, diese Serumbestandteile abzusättigen und so eine Bindung an die Beads zu verhindern. Die Inkubationsvarianten führten weder zu einer Minimierung der unspezifischen Bindung noch zu einer verbesserten Differenzierung PCV2-positiver und negativer Seren. Die in der vorliegenden Arbeit verwendeten Bead-Arten sind für den Nachweis von Antikörpern gegen das PCV2 ORF2-Protein nicht geeignet. Alternative Bead-Arten für einen vergleichbaren Versuchsansatz stehen derzeit nicht zur Verfügung. Ein weiteres Ziel der Arbeit bestand darin, eine bereits in der Diagnostik von Schweineviren etablierte Methode, die PCR, zu einer Multiplex-PCR zu erweitern. Als zu detektierende Parameter wurden die derzeit bedeutendsten viralen Erreger von respiratorischen Erkrankungen des Schweins, das PRRS-Virus (Typ 1 und Typ 2), das Porzine Influenzavirus mit den Subtypen H1N1, H3N2 und H1N2 und PCV2 gewählt. Es wurden die Primersequenzen von bereits etablierten Einfach-PCRs an die besonderen Ansprüche einer Multiplex-PCR angepasst und die Methode zunächst im Einzelansatz auf Funktionsfähigkeit überprüft. Im Anschluss wurden die Parameter zu einer Multiplex-PCR zusammengeführt, die Methode optimiert und auf Spezifität, Sensitivität und Verhalten in der Routinediagnostik überprüft. Aufgrund der im Gegensatz zur Einfach-PCR zum Teil herabgesetzten Sensitivität ist diese Methode für Ausschlussuntersuchungen weniger geeignet. Für die Untersuchung von Probenmaterial klinisch erkrankter Tiere ist sie jedoch gut geeignet und bietet die Möglichkeit einen schnellen Überblick über das Erregerspektrum zu erhalten. Es muss jedoch berücksichtigt werden, daß bestimmte Parameter, z.B. PCV2, die Sensitivität des Nachweises der anderen Parameter sehr deutlich herabsetzen kann. Dies ist insbesondere von Bedeutung, da PCV2-DNA in Probenmaterial von klinisch erkrankten Tieren in sehr hohen Mengen vorhanden ist und dadurch die weiteren Parameter noch zusätzlich beeinflusst werden können.

Orientador: Fernando Antonio de Ávila / Banca: Hélio José Montassier / Banca:Antonio José Piantino Ferreira / Resumo: Foram isoladas 90 estirpes de E. coli de fígados e de intestinos, de pintainhas de postura comercial, com sete dias de idade. Com o objetivo de caracterizar as estirpes isoladas, a patogenicidade das mesmas foi determinada, em inoculação "in vivo". O teste revelou 44 estirpes de alta ou de intermediária patogenicidade, que foram analisadas por PCR multiplex quanto á presença de oito genes de virulência (astA, iss, iucD, irp2, papC, tsh, vat e cva/cvi) e tiveram os sorotipos O:H identificados. Os resultados demonstraram que todas as estirpes analisadas continham pelo menos um dos oito genes pesquisados e que a maioria (93,20%) possuíam o gene iss. Foram detectados 17 perfis genéticos diferentes, sendo 15 deles com combinações de dois ou mais genes, representando 70,45% do total de estirpes analisadas. Onze sorogrupos e onze antígenos "H" foram identificados, sendo O8 (15,89%) e o H17 (23,8%) os mais frequentes. Com o objetivo de verificar a susceptibilidade das estirpes aos antimicrobianos: ampicilina, enrofloxacina, eritromicina, espectinomicina, estreptomicina, fosfomicina, kanamicina, lincomicina, norfloxacina, sulfa+trimetoprim e tetraciclina, comumente utilizados na avicultura, todas as estirpes de E. coli isoladas foram analisadas. O antimicrobiano que apresentou maior atividade antibacteriana foi a espectinomicina (92,2%) e o de menor atividade foi a lincomicina. Nenhuma das estirpes foi sensível a todos os antimicrobianos testados. Os resultados demonstraram uma diversidade de sorotipos e de genes de virulência envolvidos no quadro clínico de colibacilose estudado, como também sorogrupos que não haviam sido relatados em APEC e a alta incidência de resistência antimicrobiana / Abstract: A total of 90 strains of E. coli were isolated from the livers and intestines of seven-day-old commercial layer chicks. With the objective of characterising the isolated strains, their pathogenicity levels were determined by in vivo inoculation. These tests identified 44 strains with high or intermediate levels of pathogenicity, which were then analysed by multiplex PCR for the presence of eight virulence genes (astA, iss, iucD, irp2, papC, tsh, vat e cvi/cva) and the serotypes O:H were identified. The results demonstrated that these isolated strains contained at least one of the eight genes of interest, and the majority (93.20%) possessed the iss gene. Seventeen different genetic patterns were detected, 15 of which had combinations of two or more genes, representing 70.45% of all analysed strains. Eleven serogroups and eleven antigens "H" were identified, O8 (15.89%) and H17 (23.80%) were most frequent. Aiming to verify the susceptibility of strains to antimicrobial agents: ampicillin, enrofloxacin, erythromycin, spectinomycin, streptomycin, fosfomycin, kanamycin, lincomycin, norfloxacin, trimethoprim sulfa and tetracycline, commonly used in poultry, all strains of E. coli isolates were analyzed. The antibiotics that showed the highest antibacterial activity was spectinomycin (92.2%) and less activity was the lincomycin (100%) strains were resistant. None of the strains were sensitive to all antibiotics tested. The results showed a diversity of serotypes and virulence genes involved in the clinical study of colibacillosis, as well as serogroups that had not been reported in APEC and the high incidence of antimicrobial resistance / Mestre

Made available in DSpace on 2015-07-22T20:49:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-29. Added 1 bitstream(s) on 2015-08-11T03:26:21Z : No. of bitstreams: 1 Publico-178.pdf: 625763 bytes, checksum: bd311a8cd3f7ff010bc833233dd4c82b (MD5) / Objetivo: Desenvolvimento e aplicação de protocolos de Nested Multiplex PCR e PCR em Tempo Real para a detecção bacteriana e classificação de Gram em amostras de humor aquoso e vítreo coletadas de pacientes com suspeita clínica de endoftalmite. Métodos: Especificidade analítica foi estabelecida utilizando 31 microrganismos clinicamente importantes, 20 gram-positivos e 11 gram-negativos. Reação cruzada com DNA humano e DNA fúngico foi testada. Amostras controles de humor aquoso coletadas após facoemulsificação foram incluídas. Sensibilidade analítica para as metodologias de PCR foram determinadas utilizando diluição seriada 1:10 de DNA extraído de S. epidermidis e E. coli. As técnicas foram posteriormente aplicadas e testadas em amostras de humor aquoso e vítreo coletadas de pacientes com diagnóstico clínico de endoftalmite. Preparações comerciais de Taq DNA polimerase foram pré-tratadas com DNaseI. Resultados: Amplificação genérica do gene 16S rDNA foi positiva para todos os isolados bacterianos. Classificação de Gram pela técnica de Nested Multiplex PCR não foi possível apenas para isolados de Acinetobacter spp. Utilizando PCR Multiplex em Tempo Real, todos os isolados foram classificados por Gram, sendo que P. acnes exibiu um padrão misto de amplificação. Limite de detecção da metodologia de Nested Multiplex PCR foi de 1 fg/μl para S. epidermidis e E.coli. A sensibilidade de detecção para S. epidermidis e E. coli utilizando reação para detecção universal bacteriana por PCR em Tempo Real com SYBR Green foi de 100 fg/μl (E = 0,82 e 0,86; r2 = 0,99) e 1 pg/μl utilizando metodologia de PCR Multiplex em Tempo Real com Sondas TaqMan (E = 0,66 e 0,77; r2 = 0,99). A positividade da cultura para detecção bacteriana a partir de amostras de humor aquoso e vítreo foi de 47,6% e pelas metodologias de Nested Multiplex PCR e PCR em Tempo Real foi de 100% e 95,2% respectivamente. Entre as amostras negativas por cultura (n=10), a metodologia de Nested Multiplex PCR foi positiva para todas (100%) e PCR em Tempo Real em 90% dos casos (9/10). Entretanto, a proporção de falso-positivos pela metodologia de Nested Multiplex PCR (65,4%) foi superior aos valores obtidos por PCR em Tempo Real (7,7%). A classificação de gram foi obtida em 88,8% dos casos por Nested PCR e 100% por PCR em Tempo Real. A correlação entre a identificação clássica e classificação de Gram molecular foi 63,6% para ambas as técnicas. A identificação pelo sequenciamento dos produtos obtidos por Nested Multiplex PCR e PCR em Tempo Real apresentou correlação de 100% e 88,8%, respectivamente, quando comparada a identificação fenotípica. Conclusões: As metodologias de PCR apresentaram boa correlação quando comparadas com os resultados da cultura e a detecção passou de 47,6% por cultura para 100%, demonstrando serem testes viáveis e mais sensíveis para a caracterização laboratorial de endoftalmite. / Objective: Development and application of Nested Multiplex PCR and Real Time PCR assays for detection and Gram classification of bacteria using aqueous and vitreous humor collected from patients with suspected endophthalmitis. Methods: Analytical specificity was established using 31 clinically important pathogens, 20 gram-positive and 11 gram-negative. Specificity was also tested using human DNA and fungal DNA. Control samples of non-infected aqueous humor collected at the end of phacoemulsification surgery were included. Analytical sensitivity was determined using a 10-fold dilution of S. epidermidis and E. coli DNA. After, methodologies were tested in aqueous and vitreous humor collected from patients with clinical diagnosis of endophthalmitis. Comercial Taq polymersase preparations were DNA decontaminated using DNaseI pretreatment. Results: Universal amplification of 16S rDNA was achieved for all bacterial isolated. Nested Multiplex PCR failed only to determine the Gram status of Acinetobacter spp. Gram classification was achieved for every bacterial isolates using a Multiplex Gram-Specific TaqMan-based PCR, and only a P. acnes isolate showed a mixed signal. Limit of detection using Nested Multiplex PCR was 1 fg/μl for both S. epidermidis and E. coli. Sensitivity for detection of S. epidermidis and E. coli DNA using a SYBR Green 16S rDNA-based universal PCR was 100 fg/μl (E = 0.82 and 0.86; r2 = 0.99) and 1 pg/μl using a Multiplex Gram-Specific TaqMan-based PCR (E = 0.66 and 0.77; r2 = 0.99). Culture was positive in 47.6% of aqueous and vitreous humor analysis. Nested Multiplex PCR and Real Time PCR assays were positive in 100% and 95.2% of these cases, respectively. Among negative culture samples, Nested Multiplex PCR was positive for all (100%) and Real Time PCR assays in 90% of cases (9/10). Gram classification was completed for 88.8% and 100% samples using Nested Multiplex PCR and Real Time PCR methodologies, respectively. Correlation of 63.6% between microbiological and molecular Gram classification was observed using both molecular assays. 16S rDNA sequence-based identification using Nested Multiplex PCR and Real Time PCR products showed 100% and 88.8% correlation respectively when compared with phenotypic identification. Conclusions: Both PCR methodologies presented good correlation when compared with culture-proven results and bacterial detection was improved from 47.6%% to 100% showing to be feasible tests for laboratorial characterization of bacterial endophthalmitis. / TEDE / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-10-26Bitstream added on 2014-06-13T18:56:02Z : No. of bitstreams: 1 guastalli_eal_me_jabo.pdf: 288811 bytes, checksum: 03b7643862d7f270422c422ff91b53b9 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Foram isoladas 90 estirpes de E. coli de fígados e de intestinos, de pintainhas de postura comercial, com sete dias de idade. Com o objetivo de caracterizar as estirpes isoladas, a patogenicidade das mesmas foi determinada, em inoculação “in vivo”. O teste revelou 44 estirpes de alta ou de intermediária patogenicidade, que foram analisadas por PCR multiplex quanto á presença de oito genes de virulência (astA, iss, iucD, irp2, papC, tsh, vat e cva/cvi) e tiveram os sorotipos O:H identificados. Os resultados demonstraram que todas as estirpes analisadas continham pelo menos um dos oito genes pesquisados e que a maioria (93,20%) possuíam o gene iss. Foram detectados 17 perfis genéticos diferentes, sendo 15 deles com combinações de dois ou mais genes, representando 70,45% do total de estirpes analisadas. Onze sorogrupos e onze antígenos “H” foram identificados, sendo O8 (15,89%) e o H17 (23,8%) os mais frequentes. Com o objetivo de verificar a susceptibilidade das estirpes aos antimicrobianos: ampicilina, enrofloxacina, eritromicina, espectinomicina, estreptomicina, fosfomicina, kanamicina, lincomicina, norfloxacina, sulfa+trimetoprim e tetraciclina, comumente utilizados na avicultura, todas as estirpes de E. coli isoladas foram analisadas. O antimicrobiano que apresentou maior atividade antibacteriana foi a espectinomicina (92,2%) e o de menor atividade foi a lincomicina. Nenhuma das estirpes foi sensível a todos os antimicrobianos testados. Os resultados demonstraram uma diversidade de sorotipos e de genes de virulência envolvidos no quadro clínico de colibacilose estudado, como também sorogrupos que não haviam sido relatados em APEC e a alta incidência de resistência antimicrobiana / A total of 90 strains of E. coli were isolated from the livers and intestines of seven-day-old commercial layer chicks. With the objective of characterising the isolated strains, their pathogenicity levels were determined by in vivo inoculation. These tests identified 44 strains with high or intermediate levels of pathogenicity, which were then analysed by multiplex PCR for the presence of eight virulence genes (astA, iss, iucD, irp2, papC, tsh, vat e cvi/cva) and the serotypes O:H were identified. The results demonstrated that these isolated strains contained at least one of the eight genes of interest, and the majority (93.20%) possessed the iss gene. Seventeen different genetic patterns were detected, 15 of which had combinations of two or more genes, representing 70.45% of all analysed strains. Eleven serogroups and eleven antigens “H” were identified, O8 (15.89%) and H17 (23.80%) were most frequent. Aiming to verify the susceptibility of strains to antimicrobial agents: ampicillin, enrofloxacin, erythromycin, spectinomycin, streptomycin, fosfomycin, kanamycin, lincomycin, norfloxacin, trimethoprim sulfa and tetracycline, commonly used in poultry, all strains of E. coli isolates were analyzed. The antibiotics that showed the highest antibacterial activity was spectinomycin (92.2%) and less activity was the lincomycin (100%) strains were resistant. None of the strains were sensitive to all antibiotics tested. The results showed a diversity of serotypes and virulence genes involved in the clinical study of colibacillosis, as well as serogroups that had not been reported in APEC and the high incidence of antimicrobial resistance

Leber’s hereditary optic neuropathy (LHON) is the most commonly inherited disease that causes blindness in one or both eyes, with a minimum prevalence of 1 in 31 000 in the northeast of England. What causes LHON is not fully known but three mitochondrial mutations, G3460A, G11778A, and T14484C, have been identified in over 95 % of all LHON patients. To diagnose LHON, detection methods like sequencing, allele specific polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) are used to identify these three mutations. The methods are now evolving into multiplex ones to increase efficiency, the aim of this study was therefore to optimize one of them, a multiplex PCR-RFLP method developed in 2015. This study was however not completed, due to the COVID-19 pandemic, but a series of preparatory steps were performed before its termination. DNA extraction was performed, both genomic and plasmid, using a kit and in-house protocols. The DNA was then used for polymerase chain reactions (PCRs), for both the human β-globin gene and for the three mutations, where magnesium concentration and annealing temperature was optimized. This study resulted in clear, high quality extractions, with the kit as the preferable method. It also indicated that a 3 mM magnesium concentration and an annealing temperature of 59 °C was optimal for all mutations when using so called LHON primers. The conditions for the PCR using the multiplex primers might be different, therefore a new study is required to evaluate the multiplex PCR-RFLP method further. / Bakgrund: Lebers hereditära optikusneuropati (LHON) är en vanlig ärftlig sjukdom som orsakar blindhet. LHON orsakas i över 95 % av fallen av en av tre mitokondriella mutationer, där en byggsten i mitokondriens DNA felaktigt bytts ut mot en annan. Dessa mutationer heter G3460A, G11778A och T14484C. För att diagnostisera sjukdomen detekteras mutationerna, bland annat genom att extrahera DNA från blod, DNA som man sedan skapar otaliga kopior av genom en metod som heter ”polymerase chain reaction” (PCR). Dessa kopior kan sedan klyvas i bitar med hjälp av enzym och baserat på fragmentens storlek kan det avgöras om personen har mutationen eller inte, detta kallas för ”restriction fragment length polymorphism” (RFLP). I nuläget letar man efter en mutation i taget men det har utvecklats några metoder där man kan hitta alla mutationer på en gång och den här studiens syfte var att undersöka hur man på bästa sätt kan utföra en av dessa metoder, en så kallad multiplex PCR-RFLP. Metod: Studien avbröts i förtid på grund av ett pandemiskt utbrott av COVID-19 men hann omfatta DNA-extraktion från humant blod och bakterier med hjälp av ett kommersiellt kit och laboratoriets egna protokoll. Även PCR utfördes för en normal genuppsättning och de tre mutationerna. Resultat och slutsats: Extraktionen gav bra resultat med alla metoder men det kommersiella kitet gav bäst resultat. PCR med det DNA som extraherats fungerade bara ibland vilket gjorde det svårt att dra några större slutsatser, oavsett krävs fler studier för att undersöka metoden eftersom arbetet inte kunde slutföras.

The aim of this thesis was to determine the authenticity of fruit food for infants using molecular and instrumental methods. In the experimental part, plant DNA isolations from fruit leaves (peaches, apricots, plums and apples) and bananas were performed. Further, DNA was isolated also from five commercial products, and from model mixtures that were prepared in terms of content identical to the commercial mixtures. The isolated DNA was characterized and verified by qPCR with plant DNA-specific ITS2 primers. Three triple primer pairs were selected, and their specificity was evaluated when performing multiplex PCR. This method makes it possible to detect more types of fruit in one reaction, reducing the economic and time requirements for detection. As none of the selected primer pairs were sufficiently specific for the apricot, the evidence from the plum and peach was further realized using duplex PCR. High resolution melting curve analysis was used for better DNA type recognition. Subsequently, agarose gel electrophoresis was performed to analyse the fragment lengths. Furthermore, experiments have been made to identify some specific phenolic substances in commercial and model fruit mixtures by HPLC. Since phenolic substances are degradable under unsuitable storage conditions, the presence of individual compounds was not detected by this method.