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.

<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>

Philosophiae Doctor - PhD / This thesis has been placed under a long term embargo. Forensic and population genetic parameters were investigated in the Botswana population using autosomal and Y-chromosome short tandem repeat markers. AmpFlSTR Profiler plus markers were used to investigate the genetic diversity and forensic parameters in 773 individuals from Botswana from the reference database of the Botswana Police. The levels of polymorphism found using the AmpFlSTR Profiler Plus markers showed that the nine loci that make up the AmpFlSTR Profiler Plus can differentiate individuals for forensic casework in the Botswana population. AmpFlSTR Identifiler autosomal STR markers were used to investigate the population structure according to ethno-linguistics and geography 990 individuals from Botswana that serve as a reference database for the Botswana Police. Using pairwise genetic distances (Fst), analysis of molecular variance (AMOVA), factorial correspondence analysis (FCA), and the unsupervised Bayesian clustering method found in STRUCTURE and the landscape genetics software TESS, ethno-linguistics were found to have a greater influence on population structure than geography. The patterns of population structure found using these markers highlight the need for regional reference databases that include both ethnolinguistic and geographic location information. These markers have important potential for bio-anthropological studies as well as for forensic applications. The 17 Y-chromosomal short tandem repeats found in AmpFlSTR Y-filer and a highly discriminatory Y-STR genotyping system (the Y-STR 10-plex developed in the Forensics DNA Laboratory at the University of the Western Cape) were analysed in 249 unrelated male individuals from Botswana. Rst, multi-dimensional scaling (MDS) and AMOVA were used to investigate population differentiation in Botswana. The discrimination capacity (DC) was found to be higher using the Y-STR 10-plex as compared to the 17 markers in the Y-filer genotyping system. No geographic regional or ethnic differentiation was observed between the Northern and Southern regions of Botswana using both marker systems. Regional and ethnic variation can be useful in forensic working hypotheses. Cluster analysis using the highly discriminatory Y-STR 10-plex haplotypes may provide information about ancestry and haplogroup information. / National Research Foundation (NRF)

Os pacientes infectados pelo vírus da imunodeficiência humana (HIV) freqüentemente apresentam alterações digestivas, sendo o esôfago um alvo comum de lesões estruturais. A etiologia infecciosa é a mais freqüente neste grupo de pacientes. Múltiplos agentes já foram implicados como causadores de lesões esofágicas. As infecções virais são uma das principais causas de tais lesões, sendo os vírus mais implicados o citomegalovirus (CMV) e o vírus herpes simples (HSV). Muitas lesões ulceradas permanecem sem diagnóstico etiológico, mesmo após exaustiva investigação, sendo denominadas úlceras idiopáticas ou aftosas. Os métodos de diagnóstico usuais são demorados e pouco sensíveis. Assim, nosso estudo tem como principal objetivo estudar o papel do método da reação em cadeia da polimerase (PCR) no diagnóstico destas lesões. Durante o período de outubro de 1996 a outubro de 1997, foram estudados 79 pacientes HIV-positivos, que foram submetidos ao exame de endoscopia digestiva alta por indicação clínica. Estes foram submetidos a 89 exames endoscópicos, sendo colhidas 96 biópsias, as quais foram armazenadas em nitrogênio líquido (50) ou em freezer a –70oC (46). O DNA foi extraído usando método baseado na lise hipotônica, digestão com proteinase K, extração com fenol-clorofórmio e precipitação em etanol. Uma quantidade fixa foi usada para amplificação em ciclador térmico, utilizando primers específicos para CMV, Herpesvirus, HPV, HIV, Haemophilus ducreyi, Treponema pallidum e as micobactérias M. tuberculosis, M. avium e M. intracellulare. O produto final foi submetido a uma eletroforese em gel de agarose e corado com brometo de etídeo. A endoscopia não revelou alterações esofágicas em 26 exames (29,2%). As alterações observadas foram monilíase esofágica em 33 exames (37,1%), úlceras em 22 (24,7%); esofagite em 10 (11,2%) e áreas lugol-negativas em 9 (10,1%). A PCR resultou positiva para o CMV em 19 amostras (19,8%), para o Herpes em 4 (4,2%), para o HPV em 17 (17,7%), para o HIV em 37 (38,5%) e para o H. ducreyi em 3 (3,1%). Nenhuma amostra foi positiva para o T. pallidum e para micobactérias. No estudo de 29 amostras de 22 úlceras esofágicas a PCR detectou o CMV em 9 amostras (31%), o Herpes em 3 (10,3%), o HPV em 6 (20,7%), o HIV em 19 (65,5%) e o H. ducreyi em 2 (6,9%) e em 8 (36,4%) não foi detectado nenhum agente. O CMV foi detectado com freqüência nas úlceras esofágicas, sendo difícil diferenciar se havia infecção ativa ou latente. O HIV teve uma incidência elevada nas biópsias de úlceras, o que pode sugerir um possível papel etiológico deste agente em tais lesões. O HPV foi o terceiro agente mais freqüente, mas não foi possível caracterizá-lo como causador de lesões esofágica ulceradas. A PCR apresentou potencial para tornar-se um método útil na investigação das lesões esofágicas em pacientes infectados pelo HIV. / Patients infected by Human Immunodeficiency Virus (HIV) usually present digestive abnormalities and the esophagus is a common target of structural lesions. Infections are the most frequent cause of esophageal lesions in these patients. Several agents were already implied in this process. Viral infections are one of the main causes of such lesions and cytomegalovirus (CMV) and herpes simplex virus (HSV) were the most involved agents. Many ulcerated lesions persist without etiologic diagnosis even after exhaustive investigation, being denominated idiopathic or aphthous ulcers. The usual diagnostic methods are difficult and have low sensitivity. Thus, the main objective of our study was to evaluate the role of the polimerase chain reaction (PCR) method in the diagnosis of these lesions. During the period of October of 1996 to October of 1997, 79 HIV-positive patients were studied. They were submitted to upper digestive endoscopies, which were indicated on clinical basis. These patients were submitted to 89 upper digestive endoscopies, being obtained 96 biopsies, which were stored in liquid nitrogen or in a 70oC freezer. DNA was extracted using a method based on hypotonic lyses, proteinase K digestion, extraction with phenol-chloroform and precipitation in ethanol. A fixed amount was used for amplification in thermal cycler, using specific primers for CMV, herpesvirus, human papillomavirus (HPV), HIV, Haemophilus ducreyi, Treponema pallidum, Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare. The final products were submitted to an electrophoresis in agarose gel and stained with ethidium bromide. The endoscopies did not reveal esophageal alterations in 26 exams(29,2%). The abnormalities observed were esophageal candidiasis in 33 exams (37,1%), ulcers in 22 (24,7%); esophagitis in 10 (11,2%) and lugol-negative areas in 9 (10,1%). The PCR was positive to CMV in 19 samples (19,8%), for Herpes in 4 (4,2%), for HPV in 17 (17,7%), for HIV in 37 (38,5%) and for the H. ducreyi in 3 (3,1%). No sample was positive for T. pallidum or micobacterium. In the study of the esophageal ulcers by PCR, CMV was detected in 9 samples (31%), Herpes in 3 (10,3%), HPV in 6 (20,7%), HIV in 19 (65,5%), H. ducreyi in 2 (6,9%) and any agent was detected in 8 samples (36,4%). CMV was frequently detected in esophageal ulcers, being difficult to differentiate between active and latent infections. The HIV had an elevated incidence in ulcer biopsies, which may suggest a possible etiologic role of this virus in such lesions. HPV was the third more frequent agent, but it was not possible to attribute the esophageal lesions to that virus. In conclusion, this study suggests that the PCR can be an useful method in the investigation of esophageal lesions in HIV infected patients.

Salmonella species is the etiologic agent of salmonellosis, which is a zoonotic infection that is characterized by diarrhea and systemic infection. Contaminated foods are usually the vehicles of Salmonella transmission along the food supply chain. Asymptomatic food production animals and effluents also contribute to contamination of meat. Antimicrobials have contributed significantly to treatment of salmonellosis. However, uncontrolled antimicrobial use is among the causes of antibiotic resistance, which results in treatment failure. The aim of this research study was to determine the extent of Salmonella spp contamination during the cattle slaughtering process in South African rural abattoirs (n = 23), water and cattle feaces. In addition, the aim was to determine antimicrobial resistance profiles of the Salmonella spp isolates. The specific objectives were: i) to establish the occurrence and distribution of Salmonella spp on cattle carcasses, hides, and intestinal contents and environmental samples using classical microbiology and molecular techniques; ii) to determine the Salmonella serovars using serotyping; and iii) to determine antimicrobial resistance patterns and multidrug resistance among the Salmonella isolates using the Kirby-Bauer disc diffusion method. Materials and Classical microbiology techniques were used to analyze cattle faeces (n = 400), hides (n = 67), intestinal contents (n = 62), carcass sponges (n = 100), and water from the abattoirs (n = 75) for the presence of Salmonella spp. Further confirmation of the Salmonella isolates was done using Polymerase Chain Reaction whereby the invA gene was targeted. A total of 92 Salmonella spp isolates were recuperated. The 92 Salmonella were serotyped as described in the White-Kauffmann-Le Minor scheme. The 92 Salmonella spp isolates were further subjected to antimicrobial susceptibility examination towards the following antimicrobials: ampicillin (10μg), cefotaxine (30μg), kanamycin (30μg), oxytetracycline (30μg), and enrofloxacin (5μg) by using the Kirby-Bauer disk diffusion procedure. All the isolates carried the invA genes. The average Salmonella spp occurrence on carcasses, hides, and intestinal contents was 35.37% (n = 81). Eleven of the faecal samples (2.75%) tested positive for Salmonella spp. The Salmonella serovar that occurred more frequently was S. Enteritidis. Different serovars that were recognized on carcasses were not automatically found on the hides and intestinal contents. The incompatible frequency of the different Salmonella serovars on carcasses, intestinal contents and hides means that in addition to carriage on hides and in intestinal contents, new external causes that did not form part of this study also play a vital role concerning carcass contamination. Most Salmonella spp (n = 66; 71.7%) isolates were resistant to a minimum of one antimicrobial with main resistance detected towards oxytetracycline (51.90%). This emphasizes on the call for wise antimicrobial use at some stage in animal production and strict sanitation for the duration of slaughtering. Briefly, cattle slaughtered in South African rural abattoirs harboured different types of Salmonella serovars that were resistant to antimicrobials, which could be a public health risk and danger. The outcome should support policymakers with determining the effectiveness of existing sanitary measures during cattle slaughtering in rural abattoirs, which is vital from socio-economic, public health, and epidemiological perspectives. / College of Agriculture and Environmental Sciences

In the food industry, it is very important to take care of the quality, safety and organoleptic properties of the products supplied. For this reason, food must be checked. However, not all information can be found using conventional techniques such as immunoassays, chromatographic techniques, etc. DNA-based techniques can be used for these cases where traditional procedures are insufficient. Among them, the best known technique is PCR. The aim of the thesis was to isolate DNA from vegetable samples (broccoli, beetroot, carrot and pepper). DNA was isolated using the magnetic particle method and the traditional CTAB method. Both methods were able to isolate the DNA from the vegetable samples in quality and at a concentration suitable for PCR, where the 35S rDNA gene region was amplified (more precisely about 700 bp of the 18S-ITS1-5,8S region). After amplification, the PCR products were subjected to restriction reactions and the results compared to bioinformatic analysis. These steps have succeeded in finding suitable enzymes for diferentiation of PCR products from the tested vegetable species.

The effectiveness of magnetic microparticles in isolation of DNA from Lactobacillus rhamnosus CCM 1825T and DNA from chicken erythrocytes were studied in diploma thesis. Magnetic HEMA based microparticles coated by carboxylic groups and hyperbranched styrene-divinylbenzene particles (IMC AS ČR, Prague, Czech Republic) were used for DNA isolation. Magnetic microparticles Dynabeads® DNA DIRECT™ Universal (Dynal, Norway) based on polystyrene and MPG® Uncoated (PureBiotech, USA) based on magnetic glass were used as a control. The dependence of amount of eluted DNA on concentration of DNA in the base solution and the dependence of amount of eluted DNA on concentration of magnetic microparticles were studied. The affinity of magnetic microparticles to RNA for various concentrations of RNA solution was studied, too. The ability of tested particles to isolate DNA from real samples was validated using milk product Actimel. The quality of isolated DNA of Lactobacillus genus was proved using genus specific PCR.

Micromethod of DNA isolation using magnetic particles is one of the modern technological methods used in DNA isolation, and makes the process simpler, more effective and faster. The main aim of this study was to isolate the DNA from various plant (tomato) food products, using different types of magnetic particles. The results were compared and the quantity, purity and the possibility of amplication of the isolated DNA among samples were found to be different. The DNA isolation method using magnetic particles P(HEMA-co-GMA) or HPS B-M-NH2 was shown to be the most effective in achieving the above mentiond parametres. DNAs from the analysed samples of plant food products were isolated in sufficient quantity and quality to be used in the conventional PCR. Differences in the possibility of the amplification of the isolated DNA stored at -20 °C during more than a half year were not found.

INTRODUÇÃO: A síndrome de Williams-Beuren (SWB; OMIM #194050) é causada por microdeleções em hemizigose de genes contíguos na região 7q11.23. Fácies típico, estenose aórtica supravalvar (EASV), deficiência intelectual, personalidade amigável e hiperacusia constituem as principais características da SWB. A deleção mais freqüente é de 1,55Mb, porém deleção de 1,84Mb também já foi descrita. Embora a técnica de hibridização in situ por fluorescência (FISH) ser amplamente utilizada na detecção da deleção, os marcadores microssatélites são considerados altamente informativos e de fácil manejo. OBJETIVOS: Testar os marcadores microssatélites para o diagnóstico da SWB, determinar o tamanho e origem parental da microdeleção, comparar as características clínicas entre os pacientes com diferentes tamanhos da deleção e origem parental. MÉTODOS: Foram estudados 97 pacientes com diagnóstico clínico de SWB utilizando cinco marcadores microssatélites: D7S1870, D7S489, D7S613, D7S2476 e D7S489_A. A partir do DNA genômico dos probandos e de seus genitores, foi realizada reação em cadeia da polimerase (PCR), seguida de eletroforese em gel de poliacrilamida desnaturante (uréia, com 7,5 M) e os resultados visualizados com coloração de prata. RESULTADOS E DISCUSSÃO: Com cinco marcadores juntos, o resultado foi informativo em todos os pacientes. O marcador mais informativo foi D7S1870 (78,4%), seguido por D7S613 (75,3%), D7S489 (70,1%) e D7S2476 (62,9%). A deleção foi encontrada em 84 (86,6%) pacientes e sua ausência em 13 (13,4%) pacientes. A deleção de 1,55 Mb foi observada em 76/84 pacientes (90,5%) e 1,84 Mb em 8/84 pacientes (9,5%). A origem parental da deleção foi materna em 44/84 pacientes (52,4%) e paterna em 40/84 pacientes (47,6%). Alterações oculares foram mais freqüentes em pacientes com deleção. Não houve diferença nos achados clínicos entre o tamanho ou a origem parental da deleção, exceto para EASV, presente em maior freqüência nos pacientes com deleção paterna. Os resultados por marcadores microssatélites foram concordantes com FISH positivos, no entanto, em dois pacientes com FISH negativos, a microdeleção foi detectada apenas pelos marcadores. CONCLUSÃO: O uso de cinco marcadores microssatélites selecionados foi informativo em todos os pacientes. Em dois casos, os marcadores foram mais eficientes que FISH, portanto pode ser considerado um método alternativo para o diagnóstico molecular da SWB / INTRODUCTION: Williams-Beuren syndrome (WBS; OMIM 194050) is caused by hemizygous contiguous gene microdeletions at 7q11.23. Typical facies, supravalvular aortic stenosis (SVAS), mental retardation, overfriendliness and hiperacusis comprise typical symptoms in WBS. The most common deletion is 1.55 Mb, however 1.84 Mb deletion also has been described. Although fluorescence in situ hybridization (FISH) is widely used in the detection of deletion, microsatellite markers are considered highly informative and easily manageable. PURPOSE: Test the microsatellite markers for the diagnosis of WBS, to determine the size and parental origin of microdeletion, compare the clinical characteristics between patients with different sizes of the deletion and parental origin. METHODS: We studied 97 patients with clinical diagnosis of WBS using five microsatellite markers: D7S1870, D7S489, D7S613, D7S2476 and D7S489_A. From the genomic DNA of probands and their parents was performed polymerase chain reaction (PCR), followed by electrophoresis on denaturing polyacrylamide gel (urea, 7.5 M) and the results visualized with silver staining. RESULTS AND DISCUSSION: Using five markers together, the result was informative in all patients. The most informative marker was D7S1870 (78.4%) followed by D7S613 (75.3%), D7S489 (70.1%) and D7S2476 (62.9%). The deletion was found in 84 (86.6%) patients and absent in 13 (13.4%) patients. The deletion of 1.55 Mb was observed in 76/84 patients (90.5%) and 1.84 Mb in 8 / 84 patients (9.5%). The parental origin was maternal in 44/84 patients (52.4%) and paternal in 40/84 patients (47.6%). Abnormalities ocular were more frequent in the patients with a deletion. There were no clinical differences in relation to either the size or parental origin of the deletion, except for SVAS, present in more frequency in the patients with paternal deletion. The results for microsatellite markers were concordant with FISH positive, however, in two patients with negative FISH, the microdeletion was detected only by the markers. CONCLUSION: Using these five selected microsatellite markers was informative in all patients. In two cases, the markers were more efficient than FISH, thus can be considered an alternative method for molecular diagnosis in SWB

Made available in DSpace on 2015-04-11T13:38:17Z (GMT). No. of bitstreams: 1 Norma Suely de Lima Freitas.pdf: 2299430 bytes, checksum: 139bbbb8dc0c714ef7d443c796111a50 (MD5) Previous issue date: 2007-02-28 / Fundação de Amparo à Pesquisa do Estado do Amazonas / Chlamydia trachomatis is a sexually transmitting bacterium that causes great impact on females reproductive health and also, constitutes an important problem for the publichealth. The infection by C. trachomatis is estimated about 90 million cases worldwide. C. trachomatis is considerated the most prevalent sexually transmitting bacterium mainly in developing countries and causes diseases on urogenital tract, venereal limphogranuloma and others. One of the risks of infection is the practice without protection among adolescents. The recurring risk is common without the use of preservatives. The diagnostic is critical because most of the times the infection is assynptomatic. In females, the infection by C. trachomatis cause pelvic inflammatory disease (PID) and the consequences can lead to infertility, ectopic pregnancy and chronicle pelvic pain. The nucleic acid amplification techniques allow us to use small amount of samples to detect Chlamydia. The choice of the technique to detect Chlamydia trachomatis was Polymerase Chain Reaction (PCR) that offers greater sensitivity and specificity than tests as immunoassay and bacteria culture, for estimate the prevalence of this pathogen in endocervical samples of infertiles women attended in the clinic of infertility of the Dona Francisca Mendes University Hospital, in Manaus-Am-Brazil. The study population consisted of 106 women with infertility diagnosis and was realized medical gynecologic examination to obtain samples for the amplification of Chlamydia trachomatis DNA plasmid. Informations of the socialeconomic-demographs variations and clinics variations were obtained through questionaire and through signature consent term that were aplicated for each patient that participated in this study. For the statistical analysis were used the software Epi-Info 3.3 for Windows and the significant level used in the tests were 5%. The prevalence found for Chlamydial infection by the PCR method were 52,8%. To confirm the weak band found of 241 pb at the amplification region, were realized the analysis on the 6% Poliacrilamid gel and DNA sequencing of DNA Chlamydial plasmid. In relation to the PCR test and socialdemographs variations, the statistical analysis demonstrated significant assocation of 5% (p < 0,05) only for the family income (2 to 4 salaries). About variability of genes, were verified at ten samples that were sequenced minimal variations from 1,1% up to 3,3% comparing to the Chlamydia trachomatis plasmid. Due to the high prevalence found of Chlamydia trachomatis in this study, were verified the necessity to implant the detections programs in large scale, using the PCR method in clinicals samples, for having the most sensitive and specificity to determine the reduction of this pathogen in sexually active men and women. / A Chlamydia trachomatis é uma bactéria sexualmente transmissível, de grande impacto no sistema reprodutivo das mulheres, sendo também um importante problema para a Saúde Pública. A estimativa dos casos de infecção por C. trachomatis é de 90 milhões em todo o mundo. A C. trachomatis é considerada a bactéria sexualmente transmissível de maior prevalência, principalmente em países desenvolvidos e causa doenças do trato urogenital, linfogranuloma venéreo (LGV) e outras. Um dos fatores de risco para a infecção é a prática sexual sem proteção que é comum em adolescentes. O risco da recorrência sem uso do preservativo é comum. O diagnóstico é crítico devido à freqüência de infecções assintomáticas. Em mulheres, a infecção pela C. trachomatis causa doença inflamatória pélvica (DIP) e as suas conseqüências podem ser a infertilidade, gravidez ectópica e dor pélvica crônica. As técnicas de amplificação de ácidos nucléicos permitem utilizar pequenas quantidades de amostras para a detecção da clamídia. A escolha da técnica para detecção de Chlamydia trachomatis foi Reação em Cadeia de Polimerase (PCR) que apresenta uma maior sensibilidade e especificidade do que os testes de imunodetecção e de cultura celular, para estimar a prevalência desse patógeno em amostras endocervicais de mulheres inférteis atendidas na clínica de infertilidade do Hospital Universitário Dona Francisca Mendes, na cidade de Manaus-AM-Brasil. A população de estudo consistiu de 106 mulheres com diagnóstico de infertilidade e foi realizado o exame médico ginecológico para a obtenção de amostras para o exame de amplificação do DNA plasmidial da Chlamydia trachomatis. As informações das variáveis sócio-econômico-demográficas e variáveis clínicas foram obtidas através de questionário, mediante da assinatura do termo de consentimento livre e esclarecido aplicado para cada paciente que participou do estudo. Para a análise estatística foi utilizado o software Epi-Info 3.3 para Windows e o nível de significância utilizado nos testes foi de 5%. A prevalência por infecção clamidial encontrada pelo método de PCR foi 52,8%. Para confirmação das bandas fracas de 241 pb encontradas na região amplificada, realizou-se a análise no gel de Poliacrilamida 6% e sequenciamento do DNA plasmidial de Chlamydia trachomatis. Com relação ao teste da PCR e variáveis sócio-demográficas, a análise estatística realizada demonstrou associação significativa de 5% (p < 0,05) apenas para renda familiar (2 a 4 salários mínimos). Quanto à variabilidade gênica verificou-se que nas dez amostras seqüenciadas houve mínima variabilidade genética variando de 1,1% a 3,3% em relação ao plasmídio de Chlamydia trachomatis. Devido a alta prevalência de Chlamydia trachomatis encontrada neste estudo, verificou-se a necessidade de implantação de programas de detecção em massa, utilizandose o método da PCR em amostras clínicas, por possuir maior sensibilidade e especificidade para determinar a diminuição na incidência deste patógeno em homens e mulheres sexualmente ativas.