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O desempenho de seis métodos de extração de DNA de oocistos de Cryptosporidium spp. em fezes foram avaliados pela nested PCR do gene SSU rRNA. Os métodos consistem na combinação com pequenas variações de duas técnicas para a liberação de esporozoítos (indução de excistamento/E ou choque térmico/C) e três técnicas de purificação de DNA (fenol-clorofórmio/F, GuSCN-sílica/S ou kit QIAmp DNA Stool Mini/K). Para a avaliação da sensibilidade analítica, os testes foram realizados a partir de diluições seriadas de oocistos purificados, na ausência e na presença de 100 μl de fezes bovinas. A sensibilidade diagnóstica foi avaliada pela comparação com o método microscópico de centrífugo-flutuação em sacarose (padrão-ouro) em 15 amostras fecais de diferentes hospedeiros naturalmente infectados. Na ausência de fezes, foram avaliados apenas os métodos EF, ES, CF e CS, sendo que EF apresentou sensibilidade analítica superior, possibilitando a detecção de até 1 oocisto. Na presença de fezes, os métodos EF, EK e CK apresentaram desempenhos equivalentes, com sensibilidade de 104 oocistos. As maiores sensibilidades diagnósticas foram obtidas pelos métodos EK e CK, que possibilitaram a detecção de 13 (86,7%) das 15 amostras. Devido à alta sensibilidade analítica de EF em amostras purificadas, avaliou-se sua sensibilidade diagnóstica em amostras de oocistos purificados pelo método de centrífugo-flutuacão em sacarose, obtendo-se 100% de detecção. A presença de inibidores nas amostras fecais reduziu fortemente a sensibilidade das reações de PCR a partir de DNA extraído pelos métodos avaliados, sendo recomendável o emprego de técnicas de purificação de oocistos previamente à extração de DNA. Os resultados apontam para uma melhor eficiência dos métodos de indução de excistamento e kit QIAmp DNA Stool Mini. / The performance of six methods for DNA extraction from Cryptosporidium spp. oocysts in feces were evaluated by nested PCR of SSU rRNA gene. The methods are the combination with small variations of two techniques for the release of sporozoites (induction of excystation/E or thermal shock/C) and three techniques for DNA purification (phenol-chloroform/F, GuSCN-silica/S or QIAmp DNA Stool Mini kit/K). For the evaluation of analytical sensitivity, tests were made from serial dilutions of purified oocysts, in absence and in presence of 100 μl of cattle feces. The diagnostic sensitivity was assessed by comparison with the microscopic method of centrifugalflotation in sucrose (gold standard) in 15 fecal samples from different naturally infected hosts. In the absence of feces, only methods EF, ES, FC, and CS were tested. EF had the highest analytical sensitivity, enabling the detection of up to 1 oocyst. In the presence of feces, methods EF, EK, and CK showed similar performance, with sensitivity of 104 oocysts. The highest sensitivities were obtained by methods EK and CK, which enabled the detection of 13 (86.7%) of 15 samples. Due to the high analytical sensitivity of EF in purified samples, its diagnostic sensitivity was evaluated in samples of purified oocysts by the method of centrifugal-flotation in sucrose, resulting in 100% detection. The presence of inhibitors in fecal samples greatly reduced the sensitivity of the PCR reactions from DNA extraction methods evaluated, and the use of techniques for purification of oocysts prior to DNA extraction is recommended. The results show a better performance of the methods of induction of excystation and QIAmp DNA Stool Mini kit.

Made available in DSpace on 2016-08-10T10:38:39Z (GMT). No. of bitstreams: 1 Ian Marques Candido.pdf: 1079357 bytes, checksum: dac0a34c413d6875a597e0f2ba2a41f6 (MD5) Previous issue date: 2013-02-08 / The identification of human remains in decomposition, charred, skeletal remains and mass disasters can be performed by Forensic Genetics and, in most cases, bones and teeth are the only viable source for DNA typing .Thus, considering the large number of bones used in human identification and the need for standardization of DNA extraction in this kind of sample, the aim of this study is to compare two techniques of DNA extraction, with the possibility of automation. The analysis was performed on twenty five human bones evaluating the quantity of the extracted genetic material, genetic profiles obtained for each sample and the time analysis by method used. With magnetic bead in platform automated, analysis time was 3 hours to process 12 samples, whereas by silica column four samples in 27 hours. Magnetic bead recovered a larger amount of DNA in 88% of samples. 68% of the samples magnetic particle had a high amplification partial (9/16 loci) and silica column only 36%. Therefore, the method used magnetic bead is suitable for automating the extraction processes. / A identificação humana de restos mortais em avançado estado de decomposição, carbonizados, desastres em massa e esqueletizados pode ser realizada pela Genética Forense e, na maioria das vezes, ossos e dentes são as únicas fontes de DNA viáveis para análise. Dessa maneira, considerando o grande número de ossos utilizados na identificação humana e a necessidade de padronização da técnica de extração de DNA nesse tipo de amostra, o objetivo do presente trabalho é comparar duas técnicas de extração de DNA, com possibilidade de automação. A análise foi realizada em vinte e cinco ossos humanos avaliando a quantidade do material genético extraído, os perfis genéticos obtidos em cada amostra e o tempo de análise gasto pela metodologia de extração. Com a metodologia de extração por partículas magnéticas utilizando plataforma automatizada, o tempo de análise foi de 3 horas para processar 12 amostras, enquanto que por coluna de sílica 4 amostras em 27 horas. Partícula magnética recuperou uma maior quantidade de DNA em 88% das amostras. 68% das amostras extraídas por partículas magnéticas tiveram uma amplificação parcial alta (9/16 loci) e por coluna de sílica apenas 36%. Por conseguinte, a metodologia de extração por partículas magnéticas é apropriada para a automatização dos processos de extração.

The population genetic structure of Atlantic yellowfin tuna (Thunnus albacares) has received little attention despite the substantial fishing mortality of juveniles caused by purse seining around fish aggregating devices in the Gulf of Guinea targeting multi-species schools that also include similarly sized skipjack tuna (Katsuwonus pelamis) and bigeye tuna (T. obesus). We used sequence data from 355 bp of the mitochondrial control region I as well as six microsatellite loci to examine: (1) population structure, and (2) to look for evidence of reproductive variance. We analyzed two samples of adults from the Gulf of Mexico (GOM) and one sample of early juveniles (20-50 mm) from the Gulf of Guinea (GOG). We found no evidence of geographic or temporal differentiation among the samples. Accordingly, the null hypothesis of panmixia for yellowfin tuna in the Atlantic Ocean could not be rejected. A sudden expansion analysis based on mtDNA control region I sequence data of yellowfin tuna was highly significant. Time estimates for expansion were between 40,000 and 80,000 years before present. The associated high levels of homoplasy could be masking any existing population structure. Additional sampling from additional locations and across several years will be needed to test the hypothesis of panmixia. We also provide preliminary evidence of the Allendorf-Phelps effect, which may contribute to reproductive variance. This is the first evidence of this effect in any other tuna or pelagic species. Data indicates that early juveniles sharing the same mtDNA control region I haplotype were caught in the same tow and had a significant probability of halfsibship status as calculated from their haplotype and genotype at one microsatellite locus through kinship analysis. Sampling throughout the spawning season and across several years, as well as analysis with additional microsatellite loci that have a more even distribution of alleles, will be needed to more fully identify the sibling status of larvae and early juveniles caught in the same tow as well as the extent of this reproductive variance.

The population genetic structure of Atlantic yellowfin tuna (Thunnus albacares) has received little attention despite the substantial fishing mortality of juveniles caused by purse seining around fish aggregating devices in the Gulf of Guinea targeting multi-species schools that also include similarly sized skipjack tuna (Katsuwonus pelamis) and bigeye tuna (T. obesus). We used sequence data from 355 bp of the mitochondrial control region I as well as six microsatellite loci to examine: (1) population structure, and (2) to look for evidence of reproductive variance. We analyzed two samples of adults from the Gulf of Mexico (GOM) and one sample of early juveniles (20-50 mm) from the Gulf of Guinea (GOG). We found no evidence of geographic or temporal differentiation among the samples. Accordingly, the null hypothesis of panmixia for yellowfin tuna in the Atlantic Ocean could not be rejected. A sudden expansion analysis based on mtDNA control region I sequence data of yellowfin tuna was highly significant. Time estimates for expansion were between 40,000 and 80,000 years before present. The associated high levels of homoplasy could be masking any existing population structure. Additional sampling from additional locations and across several years will be needed to test the hypothesis of panmixia. We also provide preliminary evidence of the Allendorf-Phelps effect, which may contribute to reproductive variance. This is the first evidence of this effect in any other tuna or pelagic species. Data indicates that early juveniles sharing the same mtDNA control region I haplotype were caught in the same tow and had a significant probability of halfsibship status as calculated from their haplotype and genotype at one microsatellite locus through kinship analysis. Sampling throughout the spawning season and across several years, as well as analysis with additional microsatellite loci that have a more even distribution of alleles, will be needed to more fully identify the sibling status of larvae and early juveniles caught in the same tow as well as the extent of this reproductive variance.

Conductive paper materials consisting of conductive polymers and cellulose are promising for high-tech applications (energy storage and biosciences) due to outstanding aspects of environmental friendliness, mechanical flexibility, electrical conductivity and efficient electroactive behavior. Recently, a conductive composite paper material was developed by covering the individual nanofibers of cellulose from the green algae Cladophora with a polypyrrole (PPy) layer. The PPy-Cladophora cellulose composite paper is featured with high surface area (80 m2 g-1), electronic conductivity (~2 S cm-1), thin conductive layer (~50 nm) and easily up-scalable manufacturing process. This doctoral thesis reports the development of the PPy-Cladophora composite as an electrode material in electrochemically controlled solid phase ion-exchange of biomolecules and all-polymer based energy storage devices. First, electrochemical ion-exchange properties of the PPy-Cladophora cellulose composite were investigated in electrolytes containing three different types of anions, and it was found that smaller anions (nitrate and chloride) are more readily extracted by the composite than lager anions (p-toluene sulfonate). The influence of differently sized oxidants used during polymerization on the anion extraction capacity of the composite was also studied. The composites synthesized with two different oxidizing agents, i.e. iron (III) chloride and phosphomolybdic acid (PMo), were investigated for their ability to extract anions of different sizes. It was established that the number of absorbed ions was larger for the iron (III) chloride-synthesized sample than for the PMo-synthesized sample for all four electrolytes studied. Further, PPy-Cladophora cellulose composites have shown remarkable electrochemically controlled ion extraction capacities when investigated as a solid phase extraction material for batch-wise extraction and release of DNA oligomers. In addition, composite paper was also investigated as an electrode material in the symmetric non-metal based energy storage devices. The salt and paper based energy storage devices exhibited charge capacities (38−50 mAh g−1) with reasonable cycling stability, thereby opening new possibilities for the production of environmentally friendly, cost efficient, up-scalable and lightweight energy storage systems. Finally, micron-sized chopped carbon fibers (CCFs) were incorporated as additives to improve the charge-discharge rates of paper-based energy storage devices and to enhance the DNA release efficiency. The results showed the independent cell capacitances of ~60-70 F g-1 (upto current densities of 99 mA cm2) and also improved the efficiency of DNA release from 25 to 45%.

La tuberculose continue d’être une cause majeure de morbidité et de mortalité dans le monde, principalement dans les pays en voie de développement, bien qu’elle soit une maladie curable. Le diagnostic rapide et précis de la TB active est essentiel pour l’initiation rapide du traitement et le contrôle de la maladie. Le développement de nouveaux tests rapides de diagnostic de TB active représente un véritable challenge pour l’optimisation du diagnostic.L’objectif principal de nos travaux de thèse était de développer et d’évaluer des approches de PCR en temps réel ciblant la séquence d’insertion IS6110 pour la détection de l’ADN de MTB dans les expectorations.Nous avons tout d’abord développé une PCR en temps réel ciblant la séquence répétée IS6110 pour la quantification de l’ADN de MTB. L’évaluation des étapes d’optimisation de la sensibilité de la PCR IS6110 a permis de préciser les performances analytiques et le gain de sensibilité comparativement à une PCR ciblant le gène unique senX3. Au terme d’une comparaison de six protocoles de lyse/ extraction la méthode Chelex® s’est avérée être la plus efficace dans la récupération de l’ADN. La performance diagnostique de la PCR optimisée a été évaluée et comparée avec la PCR automatisée Xpert MTB/RIF sur un panel de 62 échantillons respiratoires.Dans un deuxième temps, nous avons comparé la performance diagnostique de la PCR IS6110 optimisée, le test Xpert MTB/RIF et la version ultrasensible récemment commercialisée du test PCR leader Xpert MTB/RIF Ultra pour la détection de l’ADN de MTB dans des expectorations ayant une faible charge bacillaire.Enfin, à partir de 203 LCR collectés dans le cadre du diagnostic de méningites aseptiques au Burkina Faso, nous avons évalué la performance de la PCR en temps réel multiplexe (IS6110, HSV1, HSV2) combinée à l’extraction par la méthode Chelex® pour la détection de l’ADN de MTB et d’Herpès. / TTuberculosis continues to be a major cause of morbidity and mortality worldwide, mainly in developing countries, despite being a curable disease. The rapid and accurate diagnosis of active TB is essential for rapid initiation of treatment and disease control. The development of new rapid diagnostic tests for active TB represents a real challenge for the optimization of the diagnosis.The main objective of our thesis work was to develop and evaluate real-time PCR approaches targeting the IS6110 insertion sequence for the detection of sputum MTB DNA. We first developed a real-time PCR targeting the IS6110 repeat sequence for the quantification of MTB DNA. The evaluation of the sensitivity optimization steps of the IS6110 PCR made it possible to specify the analytical performances and the sensitivity gain compared to a PCR targeting the single gene senX3. After a comparison of six lysis / extraction protocols, the Chelex® method proved to be the most efficient in the recovery of DNA. The diagnostic performance of optimized PCR was evaluated and compared with automated Xpert MTB / RIF PCR on a panel of 62 respiratory specimens.In a second step, we compared the diagnostic performance of the optimized IS6110 PCR, the Xpert MTB / RIF test and the highly marketed ultra-sensitive version of the Xpert MTB / RIF Ultra leader PCR assay for the detection of sputum MTB DNA. having a low bacillary load.Finally, from 203 LCR collected in the context of the diagnosis of aseptic meningitis in Burkina Faso, we evaluated the performance of the multiplexed real-time PCR (IS6110, HSV1, HSV2) combined with extraction by the Chelex® method for the detection of MTB and Herpes DNA.

O desempenho de seis métodos de extração de DNA de oocistos de Cryptosporidium spp. em fezes foram avaliados pela nested PCR do gene SSU rRNA. Os métodos consistem na combinação com pequenas variações de duas técnicas para a liberação de esporozoítos (indução de excistamento/E ou choque térmico/C) e três técnicas de purificação de DNA (fenol-clorofórmio/F, GuSCN-sílica/S ou kit QIAmp DNA Stool Mini/K). Para a avaliação da sensibilidade analítica, os testes foram realizados a partir de diluições seriadas de oocistos purificados, na ausência e na presença de 100 μl de fezes bovinas. A sensibilidade diagnóstica foi avaliada pela comparação com o método microscópico de centrífugo-flutuação em sacarose (padrão-ouro) em 15 amostras fecais de diferentes hospedeiros naturalmente infectados. Na ausência de fezes, foram avaliados apenas os métodos EF, ES, CF e CS, sendo que EF apresentou sensibilidade analítica superior, possibilitando a detecção de até 1 oocisto. Na presença de fezes, os métodos EF, EK e CK apresentaram desempenhos equivalentes, com sensibilidade de 104 oocistos. As maiores sensibilidades diagnósticas foram obtidas pelos métodos EK e CK, que possibilitaram a detecção de 13 (86,7%) das 15 amostras. Devido à alta sensibilidade analítica de EF em amostras purificadas, avaliou-se sua sensibilidade diagnóstica em amostras de oocistos purificados pelo método de centrífugo-flutuacão em sacarose, obtendo-se 100% de detecção. A presença de inibidores nas amostras fecais reduziu fortemente a sensibilidade das reações de PCR a partir de DNA extraído pelos métodos avaliados, sendo recomendável o emprego de técnicas de purificação de oocistos previamente à extração de DNA. Os resultados apontam para uma melhor eficiência dos métodos de indução de excistamento e kit QIAmp DNA Stool Mini. / The performance of six methods for DNA extraction from Cryptosporidium spp. oocysts in feces were evaluated by nested PCR of SSU rRNA gene. The methods are the combination with small variations of two techniques for the release of sporozoites (induction of excystation/E or thermal shock/C) and three techniques for DNA purification (phenol-chloroform/F, GuSCN-silica/S or QIAmp DNA Stool Mini kit/K). For the evaluation of analytical sensitivity, tests were made from serial dilutions of purified oocysts, in absence and in presence of 100 μl of cattle feces. The diagnostic sensitivity was assessed by comparison with the microscopic method of centrifugalflotation in sucrose (gold standard) in 15 fecal samples from different naturally infected hosts. In the absence of feces, only methods EF, ES, FC, and CS were tested. EF had the highest analytical sensitivity, enabling the detection of up to 1 oocyst. In the presence of feces, methods EF, EK, and CK showed similar performance, with sensitivity of 104 oocysts. The highest sensitivities were obtained by methods EK and CK, which enabled the detection of 13 (86.7%) of 15 samples. Due to the high analytical sensitivity of EF in purified samples, its diagnostic sensitivity was evaluated in samples of purified oocysts by the method of centrifugal-flotation in sucrose, resulting in 100% detection. The presence of inhibitors in fecal samples greatly reduced the sensitivity of the PCR reactions from DNA extraction methods evaluated, and the use of techniques for purification of oocysts prior to DNA extraction is recommended. The results show a better performance of the methods of induction of excystation and QIAmp DNA Stool Mini kit.

Invasive Candida infection is an increasing cause of morbidity and mortality in the immunocompromised patient. Molecular diagnosis based on genomic amplification methods, such as real time PCR, has been reported as an alternative to conventional culture for early detection of invasive candidiasis. The template DNA extraction step has been the major limitation in most reported nucleic acid based assays, due to problems in breaking fungal cell walls and incomplete purification in PCR inhibitor substances. The aim of this study was to compare enzymatic cell wall disruption using recombinant lyticase with mechanical disruption using glass beads. The QIAamp tissue kit was compared with two automated DNA extraction robots, the BioRobot M48 and NucliSens easyMAG, to determine their sensitivity, reliability and duration for DNA release of C. albicans. Mechanical cell wall disruption shortened and facilitated the extraction procedure, but the quantity of released DNA was significantly lower than when enzymatic cell wall disruption was used. Use of robots did not significantly shorten the DNA extraction time, compared with manual DNA extraction. However the NucliSens easyMAG resulted in a higher yield of target DNA compared to the BioRobot M48 and the manual QIAamp tissue kit. / Invasiva svampinfektioner är ett stort problem hos patienter med dåligt immunförsvar. Förekomst av invasiva svampinfektioner har ökat under senare år och medför hög dödlighet. En svampinfektion som inte snabbt diagnostiseras och behandlas kan bli livshotande om patientens kondition är dålig. Candida albicans är den vanligaste orsaken till invasiva svampinfektioner. Med traditionell svampidentifiering kan det ta dagar till veckor att isolera och artbestämma svampen. En snabbare metod att detektera Candida är att använda sig av molekylärbiologiska metoder som påvisar svampens arvsmassa, DNA. Svampar har en cellvägg som är svår att bryta ner och därför är DNA extraktionssteget ett av de mest rapporterade problemen vid DNA svampdiagnostik. Syftet med denna studie var att jämföra enzymatisk och mekanisk cellväggsnedbrytning av C. albicans med hjälp av enzymet lyticase respektive glaskulor. Vi jämförde också en manuell metod med två automatiska robotar för att bestämma deras känslighet, tillförlitlighet och tidsåtgång för DNA-extraktion från C. albicans. De slutsatser som nåtts är att den enzymatiska cellväggsnedbrytningen var känsligare men betydligt mer tidskrävande än den mekaniska cellväggsnedbrytningen. Denna studie visade även att en av de automatiska systemen extraherade signifikant mer DNA än den manuella metoden.

Magister Scientiae (Biodiversity and Conservation Biology) / Land-based abalone aquaculture in South Africa started in the early 1990s and is based on the local species Haliotis midae. This industry expanded with great success over the last decade. In 2006 abalone exhibiting typical clinical signs of tubercle mycosis was discovered for the first time in South African abalone culture facilities,posing a significant threat to the industry. Halioticida noduliformans, a fungus belonging to the Peronosporomycetes (formerly Oomycetes), has been identified as the causative agent of abalone tubercle mycosis (ATM). While diagnoses of this disease are currently done by gross observation and histopathology, these methods fail to be sensitive enough to identify the causative agent accurately and reliably.Molecular confirmation could provide for quicker more accurate diagnostic information. The aim of this study was to develop a DNA based molecular diagnostic test. Polymerase chain reaction (PCR) has been used to rapidly detect, characterise and identify a variety of organisms. Nucleotide sequences of the smalland large-subunit ribosomal ribonucleic acid (rRNA) and mitochondrial cytochrome oxidase subunit II (cox2) genes of H. noduliformans were compared with closely related Peronosporomycete gene sequences to identify potential PCR primer sites. H. noduliformans specific real-time quantitative PCR (Q-PCR) primer sets were designed and optimised for each of the selected genes. Results indicate that, although all tested primers sets could amplify fungal DNA, only the LSU and cox2 primer sets - v -demonstrated no cross-amplification with the closely related Peronosporomycete and non-fungal DNA tested in the present study. The H. noduliformans specific LSU primer set was chosen for further analysis and used for all subsequent real-time PCR assays. The lowest detection limit for the LSU primer set was evaluated by running Q-PCR on serial dilutions of known quantities of extracted H. noduliformans DNA.Serial dilutions were made in PCR grade water as well as in an abalone tissue matrix.The sensitivity of the Q-PCR reaction was determined to be 266 pg of H.noduliformans DNA per 25 μL reaction volume. However, inclusion of a nested PCR step, utilising universal fungal outer primers, followed by Q-PCR with the H.noduliformans LSU specific primers improved sensitivity to 0.266 pg of H.noduliformans DNA per 25 μL reaction volume. This equates to approximately 2.4spores per 25 μL reaction volume. DNA extraction protocols were optimised to ensure efficient and repeatable extraction of high quality fungal DNA from pure fungus and tissue samples spiked with known quantities of fungal DNA. PCR amplification efficiency and potential inhibition were examined for each extraction method. Results suggest that real-time PCR has great potential in monitoring and quantifying H. noduliformans on abalone culture facilities in South Africa.