Dissertations / Theses on the topic 'PCR'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'PCR.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
Jeannot, Anne-Cécile. "Diagnostic des infections grippales par PCR temps réel." Bordeaux 2, 2005. http://www.theses.fr/2005BOR2P040.
Full textRozales, Franciéli Pedrotti. "Real time-pcr e nested-pcr no diagnóstico da tuberculose pulmonar." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2013. http://hdl.handle.net/10183/72990.
Full textTuberculosis (TB) remains as an important public health problem worldwide. Therefore, the rapid detection of M. tuberculosis is of primary importance to effectively reduce transmission among patients. The aims of this study were to evaluate two molecular tests to detect M. tuberculosis complex (MTBC) directly from clinical samples. The study included 124 respiratory samples which were evaluated by two in house molecular assays for MTBC detection: Nested PCR (NPCR) and Real Time PCR (RT-PCR). The respiratory samples were also evaluated by the direct test (AFB assay). The results were compared with the results of culture and also compared with the culture results plus clinical data of patients. We used a commercial DNA sample with known quantification to establish the Limit of Detection (LOD). The LOD was 1 copy/μL for RT-PCR and 25 copies/μL for NPCR. The AFB assay presented low sensitivity – SE - (40%) and a high specificity - SP – (94%). Both molecular assays, RT-PCR and NPCR presented high SE and SP (RT-PCR 98% and 91%, NPCR 86% and 93%, respectively) compared to culture. When the results of the molecular tests were compared to the culture plus clinical data the SE and SP were 90,20% and 97,26% for RT-PCR and 80,39% and 98,63% for the NPCR, respectively. It was possible to observe a slight decrease of SE of the molecular methods in comparison to culture plus clinical data in relation to culture; however, the SP was increased, since many cases of TB could not be confirmed by culture. Furthermore we evaluated the cost of molecular assays: the NPCR cost was $17.77/test while the RT-PCR cost was $15.76/test. The RT-PCR test was faster (2 hours) than the NPCR (4 hours) to be performed. Our study confirms that PCRs may be useful for rapid diagnosis of respiratory TB, with high SP rates. It may also be very important to exclude such diagnosis, considering the high NPV found in our study. In summary, PCRs targeting IS6110 of MTB improve the accuracy of the diagnosis of pulmonary TB, with many potential positive effects for clinical management and control of the disease.
Ogorzaly, Leslie. "Intérêt du génotypage des phages ARN F-spécifiques pour estimer la pollution fécale et virale des eaux." Thesis, Nancy 1, 2009. http://www.theses.fr/2009NAN10025/document.
Full textF-specific RNA phages, which are non pathogenic viruses with similar size and structure to human enteric viruses, have been proposed like faecal pollution indicators, like models for pathogenic viruses in environment and like tools for microbial source tracking. The key trait on which the F-specific RNA phage approach of source tracking is based is that genogroups I and IV are predominantly isolated from non human faeces, while genogroups II and III are predominantly isolated from human faeces and sewage. Paradoxically, few data are available as for the genogroups distribution in environmental waters. So, the topic of this study was to provide additional information about the relationships between the genogroups of F-specific RNA phages and the level of faecal and viral pollution to environmental waters. First, the methodological work undertaken at the beginning of this project made it possible to develop the first real-time RT-PCR assays able to typing the F-specific RNA phages. The major advantages of this approach are a good sensitivity, a quick detection and a great specificity. Compared with the current tools, this method allows to avoid the phage cultivation and thus to play down the biases associated with the survival characteristics of infectious F-specific RNA phages in the environmental waters. Indeed, survival studies, realized in urban wastewater and also in ground water, have shown that the inactivation rates of infectious particles are always more important that this of viral RNA, whatever the experimental design was. Secondly, the analysis of urban wastewater samples enabled to check that F-specific RNA phages could give interesting information as for the characterization of faecal pollution in spite of the change of reference frame of measurement inherent in our detection method (genome versus infectious particle). In these kinds of samples, the majority of phages isolated belonged to the genogroups II and III, and they exhibited steady concentrations. In several particular samples, the high concentration of genogroup I phages has been associated with rainfall events and with the presence of zoonotic pathogens (Cryptosporidium and Giardia). This observation suggests the presence of an animal pollution after streaming phenomena. All the results obtained with urban wastewaters strengthen the use of F-specific RNA phages like reliable source identification tool. On the other hand, the comparison between the pathogenic virus concentrations and the results of genotyping has shown that phage genogroups are not relevant indicators for the presence of enteric viruses. For instance, norovirus and enterovirus concentrations in wastewater displayed a seasonal distribution while human genogroups exhibited steady concentrations over the time. Thirdly, two particular case studies devoted to natural waters constitute the major aspect of our work. In river water principally influenced by human wastes, genotyping results show that genogroup II is very largely isolated. For the first time, positive correlations between the concentrations of genogroup II phages, bacterial indicators (E. coli, enterococci) and human adenoviruses was observed, which attests the human faecal origin of this genogroup. Genogroup I was also often isolated but it appeared irregularly distributed. The correlation analysis has shown that genogroup I was linked neither with the concentration of genogroup II nor with that of bacterial or viral faecal indicators. The absence of a link between the concentrations of these two genogroups supports the assumption of another faecal origin. Conversely, a relationship was shown between genogroup I and the water turbidity observed at the sampling. This suggests that the origin of this genogroup could be related to streaming phenomena following precipitations. Thus, in river water the genogroup I and II would be the two most interesting genogroups in order to characterize faecal pollution. As a consequence, genogroup II/genogroup I ratio may be an interesting tool for faecal source tracking. Indeed, depending on the sign of the ratio, it seems possible to determine the main source of pollution at a given point. For example, for an E. coli concentration of 3.6log10 MPN/100mL, log-ratio values could as well be 3.8 as -1.7. With the water turbidity, this log-ratio was the only parameter enabled to highlight a change of faecal pollution nature. In ground waters protected from faecal pollution, genome of F-specific RNA phages was not observed while genome of adenoviruses was isolated in 7 samples on the 60 analyzed. This observation suggests that more persistent markers than RNA of phages could be detected in ground waters. More over, in persistence study, no degradation of adenoviral DNA was observed during all the time (200 days) of the experiment. Finally, the typing method newly developed during this study led to a better knowledge of the distribution of different the genogroups within environmental waters. F-specific RNA phage typing provides original information compared to the bacterial indicators, but does not constitute alone the universal indicator of faecal or viral pollution of waters
Fares, Fouad. "Quantification et suivi de la charge virale de l'Epstein-Barr dans le sang périphérique, par PCR-ELISA et PCR in situ : étude de la méthylation du promoteur BamHIW par PCR-enzymes de restriction." Lyon 1, 1998. http://www.theses.fr/1998LYO1T198.
Full textZiebell, Kim. "Evaluation of PCR and PCR-RFLP protocols to identify the Shiga toxins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0026/MQ51107.pdf.
Full textSPACOV, Isabel Cristina Guerra. "Utilização de marcadores de rDNA-PCR e tDNA-PCR para tipagem de isolados clínicos de Pseudomonas aeruginosa." Universidade Federal de Pernambuco, 2005. https://repositorio.ufpe.br/handle/123456789/6640.
Full textPseudomonas aeruginosa é uma bacteria Gram-negativa ubíqua e oportunista. Na rotina hospitalar, os marcadores fenotípicos nem sempre revelam a diversidade das bactérias distribuídas nos diversos setores, assim, aplicamos três métodos moleculares baseados na amplificação por PCR do locus de rDNA e tDNA para caracterizar a diversidade genética de linhagens de P. aeruginosa isoladas em um hospital público em Recife-PE, Brasil. O rDNA-PCR detectou 15% de variabilidade genética, contra 23% do tDNA-PCR e 23% do Duplex-PCR. O setor com maior diversidade genética foi a Unidade de Tratamento Intensivo do hospital, o qual apresentou quatro genótipos bacterianos diferentes. A ocorrência de linhagens de P. aeruginosa pertencentes ao mesmo genótipo e mesmo perfil de resistência a múltiplas drogas (MDR), em diferentes setores do hospital, sugere que há infecção cruzada entre pacientes. Os dados apresentados pelo rDNA-PCR, tDNA-PCR e Duplex-PCR, em associação ao perfil de susceptibilidade antimicrobiana provêem valiosas informações epidemiológicas para o controle de infecções hospitalares causadas por P. aeruginosa
Hill, Philip John. "PCR based gene engineering." Thesis, University of Nottingham, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.317040.
Full textCosta, Luciana Fachini da [UNESP]. "Avaliação comparativa entre PCR gênero-específica, PCR espécie-específica e nested PCR espécie-específica no diagnóstico da infecção por Brucella ovis." Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/94670.
Full textConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O diagnóstico da infecção por Brucella ovis em ovinos usualmente é realizado por meio de exame clínico, testes sorológicos e bacteriologia. Devido às limitações apresentadas pelas técnicas, o diagnóstico é geralmente obtido mediante aplicação de duas ou mais técnicas para obtenção de um resultado conclusivo. A Reação em Cadeia pela Polimerase (PCR) tem sido utilizada como ferramenta diagnóstica aplicável, pois é sensível, pouco dispendiosa, rápida, simples de ser realizada e permite o diagnóstico específico do agente infectante. Neste trabalho foram realizados dois experimentos. No primeiro compararam-se os percentuais de positividade em duas técnicas de PCR padronizadas, sendo a primeira gênero-específica e a seguinte espécie-específica, em 191 amostras de sêmen e 214 de urina provenientes de ovinos inoculados experimentalmente com a cepa de B. ovis REO 198. Posteriormente, desenvolveu-se a nested PCR a partir do produto amplificado da reação espécie-específica, e o percentual de positividade obtido pela nested PCR foi comparado aos obtidos pelas PCRs gênero-específica e espécie-específica. Foi observada diferença significativa no percentual de positividade (P<0,05) entre PCR gênero-específica e PCR espécie-específica (24,08% e 15,18%, respectivamente) para amostras de sêmen de ovinos e concordância moderada entre os resultados destas técnicas (kappa de 0,623); para amostras de urina, não houve diferença significativa entre as positividades obtidas pela PCR gênero-específica e a espécie-específica (10,28% e 7,011%, respectivamente), e a concordância entre os resultados foi moderada (kappa de 0,6167). A nested PCR espécie-específica apresentou percentual de positividade significativamente maior (P<0,001) quando comparada às PCRs gênero-específica e espécie-específica em amostras de sêmen (53,93%) e de urina (49,07%)...
Diagnosis of Brucella ovis infection in rams is routinely performed by clinical examination, serology and bacteriology. Due to limitations presented by each technique, the diagnosis is usually made by two or more techniques to obtain a conclusive result. The Polymerase Chain Reaction (PCR) has been used as a diagnostic tool applicable because it is sensitive, inexpensive, rapid, simple to perform and allows the specific diagnosis of infectious agents. In this study, the positivity percentages of two techniques of PCR previous described, one genus-specific and other species-specific, were measured in 191 semen and 214 urine samples from sheep experimentally infected with strain of B. ovis REO 198. Then, a species-specific nested PCR was developed from amplified products of the species-specific PCR, and the percentage of positivity obtained by nested PCR was compared to those obtained by genus and species-specific PCRs. Significant difference was observed when comparing the percentage of positivity (P <0.05) between genus-specific and species-specific PCR (24.08% and 15.18%, respectively) in semen samples from sheep, and there was moderate agreement between the results of these techniques ( kappa 0.623). In urine, no significant difference between the percentages of positives samples obtained by genus and species-specific PCR was observed (10.28% and 7.011% respectively) and the concordance between the results was moderate (kappa 0.6167). The species-specific nested PCR showed significantly higher percentage of positivity (P <0.001) when compared to genus-specific and species-specific PCRs in semen (53.93%) and urine (49.07%). Thus, according to the results obtained in this experiment, the species-specific PCR showed the lower percentage of positivity when compared to genus-specific PCR, but the implementation of the species-specific nested PCR showed highly significant increase... (Complete abstract click electronic access below)
Costa, Luciana Fachini da. "Avaliação comparativa entre PCR gênero-específica, PCR espécie-específica e nested PCR espécie-específica no diagnóstico da infecção por Brucella ovis /." Botucatu : [s.n.], 2010. http://hdl.handle.net/11449/94670.
Full textAbstract: Diagnosis of Brucella ovis infection in rams is routinely performed by clinical examination, serology and bacteriology. Due to limitations presented by each technique, the diagnosis is usually made by two or more techniques to obtain a conclusive result. The Polymerase Chain Reaction (PCR) has been used as a diagnostic tool applicable because it is sensitive, inexpensive, rapid, simple to perform and allows the specific diagnosis of infectious agents. In this study, the positivity percentages of two techniques of PCR previous described, one genus-specific and other species-specific, were measured in 191 semen and 214 urine samples from sheep experimentally infected with strain of B. ovis REO 198. Then, a species-specific nested PCR was developed from amplified products of the species-specific PCR, and the percentage of positivity obtained by nested PCR was compared to those obtained by genus and species-specific PCRs. Significant difference was observed when comparing the percentage of positivity (P <0.05) between genus-specific and species-specific PCR (24.08% and 15.18%, respectively) in semen samples from sheep, and there was moderate agreement between the results of these techniques ( kappa 0.623). In urine, no significant difference between the percentages of positives samples obtained by genus and species-specific PCR was observed (10.28% and 7.011% respectively) and the concordance between the results was moderate (kappa 0.6167). The species-specific nested PCR showed significantly higher percentage of positivity (P <0.001) when compared to genus-specific and species-specific PCRs in semen (53.93%) and urine (49.07%). Thus, according to the results obtained in this experiment, the species-specific PCR showed the lower percentage of positivity when compared to genus-specific PCR, but the implementation of the species-specific nested PCR showed highly significant increase... (Complete abstract click electronic access below)
Orientador: Jane Megid
Coorientador: Renato de Lima Santos
Banca: Sony Dimas Bicudo
Banca: Luis antônio Mathias
Mestre
Ferreira, Karin Correa Scheffer. "Detecção do vírus da raiva em órgãos de morcegos do gênero Artibeus (Leach, 1821) por meio de RT-PCR, Hemi-Nested RT-PCR e Real Time RT-PCR." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-19102012-132944/.
Full textThis study was aimed to detect the presence of rabies virus in different organs of the genus Artibeus bats using molecular techniques such as RT-PCR, hnRT-PCR, and the Real Time RT-PCR. From about 4,000 specimens of bats received for rabies diagnosis at the Pasteur Institute, 30 bats of the genus Artibeus were then selected. The selected bats presented positive results by the traditional DFA and N2A-cells inoculation test using brain tissue suspensions. Samples of salivary glands, urinary bladders, kidneys, lungs, and fecal contents and washings of the skulls were collected for the molecular techniques testing. The organs and the fecal contents were diluted at 1:10 (w/v) and the urinary bladder, at 1:20 (w/v) and these suspensions were inoculated into N2A cells for viral isolation. The extraction of the total RNA was performed by using TRIzol® and followed by the reverse transcription and the PCR and the hnRT-PCR were performed by using specific primers for the gene encoding the protein N. The product obtained by the reverse transcription technique was submitted to the Real Time RT-PCR technique, using primers and probe specific for antigenic variant 3 of the rabies virus. Of the 30 suspensions of the brain washings, 28 (93.33%) were positive in N2A cell culture inoculation, followed by the suspensions of the salivary glands (36.67%), bladders (16.67%) and fecal contents (3.33%). For the 180 samples evaluated, the results of sensitivity found for the RT-PCR, hnRT-PCR and Real Time RT-PCR techniques were 56.25%, 82.57%, and 82.19%, respectively. A comparison of hnRT-PCR and Real Time RT-PCR techniques performed by Fisher\'s exact test showed that the proportion of positives detected by the brain washings, organs and of the fecal content was non-significant (P> 0.05). Regarding the results found in hnRT-PCR and Real Time RT-PCR techniques, 100% positives were in brain washing, 90% and 93.33% in salivary glands, 83.33% and 90% in bladders, 80% and 93.33% in kidneys, 76.67% and 50% in lungs and 43.33% for both techniques on fecal contents. These results suggest that both hnRT-PCR and Real-Time PCR techniques can be used as complementary methods for the diagnosis of rabies and are sensitive enough for use in pathogenesis studies. The Real Time RT-PCR technique performed in this study proved to be faster and more sensitive and effective in detecting RABV in different organs and extra neural tissues of bats.
Benedetto, Elena. "Protocolli e applicazioni della reazione a catena della polimerasi (PCR)." Bachelor's thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amslaurea.unibo.it/9634/.
Full textDurand, Rémy. "Leishmania infantum : diagnostic par PCR et traitements vectorisés in vivo." Paris 12, 1997. http://www.theses.fr/1997PA120046.
Full textNaranjo, Luis Fabian Nuñez. "Isolamento e propagação de Astrovírus, Adenovírus, Coronavírus, Parvovírus, Rotavírus e Reovírus de aves comerciais com problemas entéricos em ovos embrionados." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/10/10133/tde-08102012-161644/.
Full textThe embryonated eggs are complex structures composed by embryo and support membranes (yolk sac, chorioallantoic membrane, amniotic sac). The embryo development and their membranes produced many kinds of cells that are necessary for successful replication of many viruses. Between the enteric viruses, the embryonated eggs have been used as the principal host for isolation, culture and propagation of many of these viruses. The present work describes the isolation, culture, propagation and macroscopic characterization of astrovírus, adenovirus, coronavirus, parvovirus, reovírus e rotavirus, related to enteric problems in commercial flows at Brazil in chicken embryonated eggs specific pathogens free (SPF). Intestinal samples from hens or chickens health or with enteric problems (diarrhea), but showed positive for chicken astrovírus, chicken parvovirus, avian nephritis vírus, avian rotavirus, avian reovirus, bronchitis infectious vírus and fowl adenovirus type 1, through PCR and RT-PRC reactions, were selected for making of isolation, culture and propagation in chicken embryonated eggs specific pathogens free (SPF). Mortality and macroscopic lesions presented in the embryos characterized by hemorrhages, edemas, stunting, enrollment, gelatin features and deformations, however the support membranes not presented any damaged. The viral confirmation in the eggs passages was carry out using the polymerase chain reaction and transcriptase reverse of the polymerase chain reaction. Chicken astrovírus, avian nephritis vírus, fowl adenovirus type 1, bronchitis infectious vírus, chicken parvovirus and avian reovírus grow well in chickens embryonated eggs specific pathogen free SPF, inoculated in the yolk sac on seven days-old, however these viruses were isolated in embryonated eggs of fourteen days-old, too. The inoculation by yolk sac resulted be a optimal route for viral isolation, getting isolate two samples of chicken parvovirus, eight samples of chicken astrovirus, seven samples of bronchitis infectious vírus, sixteen samples of avian nephritis vírus, eleven samples of fowl adenovirus type one and three samples of avian reovirus, but any samples was isolated for avian rotavirus. The macroscopic characteristics presented in the embryos were similar in all of isolated viruses. Many samples used not present lesions and were negative in the molecular detection, considering as negative samples to viral isolation. The positivity in the molecular tests (PCR and RT-PCR) and the presence of injuries in the embryo determinate the presence of enteric vírus and that chicken astrovírus, avian nephritis vírus, fowl adenovirus type 1, bronchitis infectious vírus, chicken parvovirus and avian reovírus were isolated, confirming that the chicken embryonated eggs are excellent host for isolation, culture, propagation and characterization of vírus related with enteric problems at Brazil. The patogenicity presented in the embryo determinate the harmful the vírus are and the possible damage that their will produce in the development of disease. Future works will establish the relation exist between the isolated vírus in this work and the other viruses around the world, and their roll in the enteric disease.
Silva, Sheila Oliveira de Souza. "Padronização de uma reação em cadeia pela polimerase em nested (nested-PCR) para detecção e diferenciação das espécies de Cryptosporidium spp e caracterização molecular de Cryptosporidium isolados de roedores sinantrópicos." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-24042012-151514/.
Full textCryptosporidium spp are cosmopolitan protozoan that infect fish, reptiles, amphibians, birds and mammals. More than 20 species are recognized within this genus. Rodents are groups of abundant and ubiquitous organisms that have been considered reservoirs of Cryptosporidium for infection of humans and livestock. The coding sequences of the smallest unit ribosomal (18S rRNA) of Cryptosporidium spp are characterized by intercalations amongst polymorphic and conserved regions along its 1700 base pairs. The aim of this study was to design primers specific for the 18S rRNA gene, potentially capable of amplifying any species or genotype of Cryptosporidium spp., and evaluate the attributes of the nested-PCR diagnosis based on such probes. The design of primers was performed to amplify a smaller segment as possible to maximize the sensitivity of molecular testing and preserving the discriminatory potential of the sequences amplified. The nested-PCR standardized in this study (nPCR-SH) was compared with another similar assay that has been widely used for detection and identification of Cryptosporidium spp. worldwide (nPCR-XIAO). It also aimed to characterize molecularly Cryptosporidum spp. isolated from synanthropic rodents, using these probes and targeted molecular probes. Forty five rodents were captured in public areas of the urban area of Umuarama, Paraná. The samples were subjected to three molecular tests, two targeted to gene18S rRNA (nPCR-SH and nPCR-XIAO) and another targeted to the gene encoding actin. The nPCR-SH was tested with strains of Cryptosporidum parvum, Cryptosporidum andersoni, Cryptosporidum meleagridis, Cryptosporidum hominis Cryptosporidum canis, Cryptosporidum serpentis and all were positive. Sixteen samples of rodents were positive by nPCR-SH, six by nPCR-XIAO and five by nPCR targeted to the gene encoding actin. The sequencing of the amplified fragments allowed the identification of Cryptosporidum Muris in three samples of Rattus rattus, and two new genotypes of Cryptosporidium rat genotype II and III. It was found rat genotype II in a sample of Mus musculus and genotype III in twelve samples, five from Rattus rattus and seven from Mus Musculus.The results showed that the primers designed for detection of Cryptosporidium spp in fecal samples were more efficient in amplifying regions that allow the distinction amongst the parasite species than those used in the PCR-XIAO. Cryptosporidium species or genotypes transmitted to other species such as zoonotic were not found in the studied samples, which suggest that the importance of these animals in the zoonotic transmission of cryptosporidiosis is very limited.
Abujamra, Tereza. "Detecção de agentes bacterianos envolvidos nos quadros de aerossaculite em perus através da reação em cadeia pela polimerase (PCR)." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-15122011-113034/.
Full textConsidering the increasing economic importance of exports of turkey meat and sanitary challenges that come with increased production of this species, this project proposes the detection of bacterial agents involved in airsacculitis of turkeys, through polymerase chain reaction (PCR). A total of 201 air sacs swabs were collected from turkey carcasses at a commercial slaughterhouse located in the Midwest of Brazil. These swabs were submitted to PCR for detection of Bordetela avium, Pasteurela multocida, Ornithobacterium rhinotracheale, Mycolplasma. gallisepticum, M. iowae, M. meleagridis e M. synoviae. B. avium was detected in 58 animals, representing 28.8% of the swabs analyzed. All 201 swabs were negative to detection of other six agents tested. B. avium is disseminated in Brazilians turkey herds and may have important impact in respiratory diseases in this specie under intensive production systems.
Arraes, Ana Carolina Palmeira. "Detecção da diversidade molecular de Candida spp. isoladas de UTI neonatal." reponame:Repositório Institucional da UFBA, 2013. http://www.repositorio.ufba.br/ri/handle/ri/11817.
Full textMade available in DSpace on 2013-06-10T20:55:35Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ Ana Carolina Arraes.pdf: 1876812 bytes, checksum: c4740a70b3675be50d2ebafeb0ba8fe3 (MD5)
CAPES
A incidência de candidemia tem aumentado nos últimos anos e o espectro das espécies de Candida tem mudado com a emergência das espécies não-albicans e com o aumento da resistência antifúngica. A técnica de PCR associada à análise dos polimorfismos de fragmentos de restrição (PCR-RFLP) e AP-PCR são exemplos de técnicas moleculares utilizadas para a detecção da variabilidade genética desses micro-organismos. O objetivo deste trabalho foi caracterizar molecularmente Candida spp. clinicamente importantes. Foram estudadas 82 leveduras do gênero Candida, 73 isolados clínicos e nove cepas padrão de referência da “American Type Culture Collection” (ATCC). O DNA genômico foi extraído através de lise mecânica/química e fenol-clorofórmio. Após amplificação com iniciadores ITS1 e ITS4, os produtos da PCR foram digeridos com as enzimas DdeI, HaeIII, BfaI e MspI. Outra amplificação foi realizada com o iniciador arbitrário AP-4. A identificação fenotípica revelou a frequência de 38% de C. parapsilosis, 33% de C. albicans, 27,5% de C. tropicalis e 1,5% de C. guilliermondii. Após amplificação, foi possível identificar e diferenciar as espécies C. lusitaniae, C. guilliermondii, C. famata e C. glabrata. A diferenciação entre C. albicans, C. dubliniensis C. tropicalis, C. krusei e C. parapsilosis não foi bem evidenciada com as enzimas DdeI e HaeIII. Porém, MspI conseguiu diferenciar C. tropicalis, C. krusei, C. parapsilosis, C. glabrata, C. guilliermondii, C. lusitaniae e C. famata, juntamente com BfaI que separou C. albicans de C. dubliniensis. Já a técnica de AP-PCR com o iniciador AP-4, possibilitou a distinção das nove espécies cepas padrão ATCC de Candida, pois todas apresentaram perfis de amplificação com número, intensidade e tamanho de banda específico, onde cada espécie foi alocada em grupo distinto e a relação de similaridade variou de 38-69%, ratificando o poder de diferenciação das espécies. As técnicas moleculares foram reprodutíveis e relativamente rápidas, de fácil interpretação e podem ser aplicadas na identificação de espécies clinicamente importantes de Candida para auxílio no diagnóstico mais rápido e tratamento mais eficiente.
Salvador
Jakobsson, Sanna. "Quantitative analysis of BCR-ABL1 fusion gene by Droplet Digital PCR and qRT-PCR." Thesis, Umeå universitet, Biomedicinsk laboratorievetenskap, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-103957.
Full textHipólito, Janayna Roriz. "Diagnóstico molecular para malária por nestedpcr e pcr em tempo real." Universidade Federal do Amazonas, 2006. http://tede.ufam.edu.br/handle/tede/2242.
Full textFundação de Amparo à Pesquisa do Estado do Amazonas
A malária é um problema de saúde pública na região Amazônica, mais de 200 mil casos dessa doença ocorrem anualmente no Amazonas, sendo 80% deles causados pelo P. vivax, que vem apresentando índices crescentes de morbidade, principalmente associados à diminuição da sensibilidade aos antimaláricos. Dentre as estratégias para combate e controle da doença, a identificação rápida e precisa da espécie é ferramenta indispensável para um tratamento apropriado, diminuição do risco de transmissão e melhor entendimento da epidemiologia desses parasitas. A técnica microscópica da gota espessa é a principal para diagnóstico da malária, entretanto, outros métodos vêm sendo testados, principalmente os moleculares que tem se mostrado mais sensíveis e específicos para detectar e diferenciar as espécies em baixas parasitemias. Avanços desse método, como a PCR em tempo real, permitem que o resultado do teste seja detectado simultaneamente a amplificação, diminuindo o tempo gasto para a realização do diagnóstico. Com o intuito de detectar molecularmente a malária, verificando a presença de plasmódios, o diagnóstico molecular foi realizado através das técnicas de PCR em tempo real e nested-PCR, para se fazer uma comparação desses dois métodos com o diagnóstico microscópico da gota espessa em 300 amostras criopreservadas, 200 coletadas no dia inicial do tratamento (D0), das quais 88% (176/200) eram provenientes de pacientes de Manaus, as 24 amostras restantes (12%) eram provenientes de localidades do interior do Amazonas: São Gabriel da Cachoeira (09), Tefé (08), Humaitá (04) e Careiro (03). Apenas 9% dessas amostras tinham diagnóstico microscópico de monoinfecção por P. falciparum e 91% (182/200) por P. vivax, não havia nenhuma amostra mista pelo diagnóstico microscópico. O diagnóstico molecular por nested-PCR confirmou a presença de DNA de plasmódio em 100% das amostras monoinfectadas. Adicionalmente, foram observadas infecções mistas, co-infecção de P. falciparum e P. vivax, em 19% (38/200) destas amostras. O diagnóstico molecular por PCR em tempo real (Lightcycler, Roche®) foi realizado em apenas 17% (34/200) dessas amostras. A co-positividade (sensibilidade) dos testes para P. vivax foi em média 71% e a co-negatividade (especificidade) 92%, para P. falciparum a co-positividade foi 91% e a conegatividade 79%. A concordância entre os testes foi regular. As 100 amostras restantes haviam sido coletadas no sétimo dia (D7) de tratamento e eram negativas pela microscopia. O diagnóstico molecular demonstrou 21% de positividade. Este estudo mostrou que muitas infecções mistas vêm sendo subestimadas para fins de avaliação epidemiológica, demonstrando que a sensibilidade e especificidade do diagnóstico molecular são superiores a do teste microscópico. O diagnóstico molecular seria então mais indicado como teste complementar no diagnóstico de pacientes com baixas parasitemias, na análise da quantidade de portadores assintomáticos, em estudo de infecções criptônicas e na avaliação da negativação da parasitemia para monitoramento terapêutico, e em estudos que visem a diminuição da transmissão pela existência de prováveis gametócitos persistentes após o tratamento. Entretanto, esse método não é indicado para rotina de diagnóstico de malária, uma vez que os resultados positivos por essa técnica não significam necessariamente que o paciente desenvolva a doença.
Saukkoriipi, A. (Annika). "Detection of pneumococcus by PCR." Doctoral thesis, University of Oulu, 2003. http://urn.fi/urn:isbn:9514272110.
Full textHellani, Ali. "Identification par DD-PCR de gènes testiculaires impliqués dans la spermatogenèse." Lyon 1, 1999. http://www.theses.fr/1999LYO1T159.
Full textColin, Laurence. "Elaboration d'une méthode de détection de Plasmodium vivax par PCR asymétrique." Montpellier 1, 1995. http://www.theses.fr/1995MON13510.
Full textGerbaud, Estelle Imbert-Marcille Berthe Marie. "Quantification du cytomégalovirus par la technique de PCR en temps réel." [S.l.] : [s.n.], 2003. http://theses.univ-nantes.fr/thesemed/PHgerbaud.pdf.
Full textAndresen, Dennie. "Entwicklung von Microarrays für die Multiparameteranalytik und Etablierung einer Multiplex-OnChip-PCR." Phd thesis, Universität Potsdam, 2009. http://opus.kobv.de/ubp/volltexte/2009/3946/.
Full textIn molecular diagnostics there is a need for fast and specific assay systems that could be used in the clinics and in point of care settings alike. Therefore miniaturisation and parallelisation are in the main focus of current assay development researches. The current gold standard for DNA analytics is the realtime PCR. However, this technology has its restraints in context to multiplex analysis. With the currently available technology an efficient multiplexing is only possible for four different targets per analysed sample. Microarrays in contrast offer the needed multiplex capabilities and have advanced to capable tools used in multiple fields of application. One focus of this work was the integration of Multiplex PCR and microarray technology, developing a microarray capable of analysing multiple parameters in one given sample, circumventing the problems and restraints of the exsisting technologies. As an example microarray assays for two different application fields were developed. One microarray assay for the detection of pathogens in poultry and another microarray assay for the detection of potentially allergenic food ingredients. Single- and Multiplex OnChip-PCR assays for both applications were developed and tested. OnChip-PCRs developed in this work showed high specificity in Single- and Multiplex-OnChip amplifications. The sensitivity was in the range of 10 DNA copies or 10ppm respectively for Single-OnChip-PCR in experiments for the detection of allergenic food contaminations. In Multiplex-OnChip-PCR experiments 100 DNA copies or 100ppm of food contaminents could be detected in different food matrices. A further focus of this work was the adaption of the OnChip amplification for the use in Point of Care settings. Isothermal amplification is a promising approach having the advantage of avoiding the thermocycling needed in the PCR. This opens up certain opportunities for the development of smaller, more flexible mobile diagnostic analysis devices. In this work we have evaluated the helicase dependent amplification (HDA) in terms of usability in OnChip amplification. In this work it was shown for the first time that HDA could be used for the detection of different pathogens in an Duplex-OnChip-PCR, showing the potential of this technology for integration in Point of Care settings.
Beauchamps, Patrick. "Contribution de l'amplification génique (PCR) au diagnostic de la toxoplasmose : intérêts de la PCR quantitative." Lille 1, 1999. https://pepite-depot.univ-lille.fr/LIBRE/Th_Num/1999/50376-1999-413.pdf.
Full textSaito, Cristiane Pereira Borges. "Analise do padrão de metilação da região promotora do gene humano PAX9 (-947 - +251) e analise in vitro da sua atividade transcricional." [s.n.], 2009. http://repositorio.unicamp.br/jspui/handle/REPOSIP/290013.
Full textTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-12T22:28:51Z (GMT). No. of bitstreams: 1 Saito_CristianePereiraBorges_D.pdf: 2404829 bytes, checksum: ef69d02f749dac4b0974b53ad416de6d (MD5) Previous issue date: 2009
Resumo: O gene PAX9 é um regulador chave durante o processo de desenvolvimento dentário e, particularmente em humanos, mutações neste gene estão associadas a fenótipos peculiares como agenesias dentárias. O objetivo deste estudo foi descobrir novas coordenadas acerca da atividade transcricional do promotor desse gene e de um possível padrão de metilação. Quanto ao estudo da metilação, foram avaliadas amostras de DNA genômico extraído da papila dentária de embriões humanos através de enzimas de restrição sensíveis à metilação e através da amplificação por Reação da Polimerase em Cadeia-PCR com oligos específicos. A extração de DNA de amostras incluídas em parafina foi padronizada no laboratório de Morfologia da Faculdade de Odontologia de Piracicaba, FOP-UNICAMP. Amostras de DNA extraídas de saliva humana foram usadas como controle. Para os ensaios de transcrição, foram amplificados através de ensaios com a transcriptase reversa, transcritos do gene Pax9 de Células Embrionárias de Rato, bem como de Células Odontoblastóides de Camundongo- MDPC-23 (Mouse Odontoblast Cell-like-23). Esses transcritos foram quantificados através de PCR semi-quantitativa. A região promotora do gene PAX9 humano íntegra (-947 - +251) e deletada (-485 - +22), recombinada com o vetor de expressão pGL3Basic, contendo o gene repórter luciferase após a região promotora, foi transfectada em cultura de Células Embrionárias de Rato. Todas as placas de cultura foram submetidas à ação de duas drogas: Ácido Retinóico (AR) e Dexametasona (Dex). Após a lise das células, os níveis relativos de expressão da proteína luciferase foram analisados, utilizando o kit Dual-Glo Luciferase (Promega), em um luminômetro. Os resultados mostram que: (1) O gene PAX9 encontra-se metilado in vitro; (2) O AR inibiu a síntese de RNA mensageiro transcrito pelas Células Embrionárias Rato. O promotor do gene humano PAX9 clivado nos sítios -485 e +22 e que não continha 507pb não foi afetado pelos receptores do AR. O Promotor PAX9 íntegro foi ativado na presença do AR na maior concentração da droga ; (3) A Dex estimulou a transcrição do gene Pax9 no grupo de células MDPC-23 e influenciou positivamente ambas as versões do promotor do gene PAX9 com as concentrações: 0.1µM e 1nM. Esses resultados demonstraram que os receptores dos hormônios esteróides AR e Dex podem se ligar diretamente a sequências do gene Pax9 em ratos e camundongos e que a região contendo 507pb retirada do promotor do gene PAX9 humano pode conter sítios de ligação para receptores do AR. Além disso, o sítio de metilação encontrado na região promotora do gene PAX9 humano resultou em novas perspectivas acerca do seu padrão de funcionamento em células normais.
Abstract: PAX9 is a key regulator during tooth development and plays an essential role in the patterning of murine and human dentition. In humans, mutations in PAX9 are associated with unique phenotypes of familial tooth agenesis that mainly involve posterior teeth. The objectives of this study were to gain new insights into the transcriptional activity and DNA methylation within the promoter region of human PAX9 gene in vitro. The methylation pattern was examined by studying PAX9 gene promoter in Dental Papilla of human Embryos through methylation-sensitive restriction enzyme and PCR amplified with specific oligos. DNA extractions from paraffin-embedded tissue sections were well established in Morphology Laboratory, Piracicaba Dental School. DNA samples from Buccal Epithelial Cells were used as control. In the present study, we have PCR amplified cDNAs encoding Rat Pax9 and Mouse Pax9 from Primary embryonic cell culture obtained from 13 day-old Wistar Rat and Mouse odontoblast cell-like culture-23 (MDPC-23) respectively and quantified by Semi-quantitative PCR technique. Furthermore, we examined the transcriptional activity of human Pax9 gene promoter: (-947 - +251) full Pax9 promoter and the promoter lacking 507bp (-485 - +22) through recombination into pGL3Basic expression vector and transfection in primary rat embryonic cells. Cell cultures were all submitted to selective regulation of both drugs: Retinoic Acid (RA) and Dexamethasone (Dex). Relative luciferase expression units were obtained by dual luciferase assay kit (Promega). Our results showed that: (1) human PAX9 promoter region is methylated in vitro; (2) RA inhibited Pax9 mRNA synthesis in Primary Rat embryonic cells while PAX9 promoter lacking 507bp (-485 - +22) was not activated by RA receptors. Human PAX9 full promoter activation was improved by RA treatment at the greater concentration; (3) Dex stimulated Pax9 mRNA activity in MDPC-23 and influenced positively both PAX9 promoter versions with 0.1µM and 1nM concentrations. The present experiments suggest that a 507bp region in PAX9 promoter may contain binding sites for RA receptors and that Dex and RA steroid hormone receptors may be directly bind to the sequences of murine Pax9 gene. The methylation pattern finding give rise to new perspectives on PAX9 patterning in normal cells phisiology.
Doutorado
Histologia e Embriologia
Doutor em Biologia Buco-Dental
Faganello, Fernanda de Sillos. "Validação de protocolo para detecção de Guignardia citricarpa em citros por PCR convencional e PCR em tempo real." Universidade Federal de Goiás, 2013. http://repositorio.bc.ufg.br/tede/handle/tede/4005.
Full textApproved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-01-29T19:56:16Z (GMT) No. of bitstreams: 2 Dissertação - Fernanda Bueno Sampaio - 2013.pdf: 855102 bytes, checksum: 072ad37c90c900b69a5b7d188b872f7c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5)
Made available in DSpace on 2015-01-29T19:56:16Z (GMT). No. of bitstreams: 2 Dissertação - Fernanda Bueno Sampaio - 2013.pdf: 855102 bytes, checksum: 072ad37c90c900b69a5b7d188b872f7c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-11-21
Citrus Black Spot (CBS) caused by Guignardia citricarpa is classified as quarantine disease, imposing restrictions to fresh fruits shipping to the European Union countries. In the state of Goiás, despite systematic phytosanitary surveys, its distribution and occurrence are unknown due to lack of available technologies for diagnosis. The occurrence of latent infections, the presence of an endophytic species morphologically similar to G. citricarpa, as well as time consuming for diagnosis through conventional methods require the validation of fast, efficient, reproducible, safe and sensitive methods of diagnosis to provide reliability of diagnosis and disease surveys for its detection and delimitation. Modifications of the “Cationic hexadecyl trimethyl ammonium bromide” method (CTAB) to extract DNA of G. citricarpafrom symptomatic tissues with validation of chemical purity, structural integrity, and absence of DNA inhibiting substances, for further use in diagnosis by PCR were tested. There was no difference in the average of DNA extracted for hygienized and non-hygienized tissues (328.62 ng µL -1 and 322.79 ng µL -1 , respectively), with low concentration of proteinsin the DNA solution. The extractor of DNA in amount (>300 ng µL -1 ) and quality (structural integrity) was sufficient to perform the conventional and real-time PCR analyses. The absence of inhibitors was demonstrated by real-time PCR, adding 0.1 % genetically modified standard corn DNA (event MON 810 standard ERM®-BF413b), with the amplification of the specific region of this event in all test samples. The modified CTAB method sowed repeatability and partial reproducibility among the limits acceptablein the methodology with coefficient of variability lower than 30 %. To comply with the ISO/TEC 17025:2005 norm, the method for the diagnosis of the fungus G. citricarpaby PCR conventional and real time PCR was validated with the specific evaluation of the limitof detection. Conventional and real time PCR methods were specificity and adequacy to detect G. citricarpa. Conventional PCR presented detecting limit of 10 ng µL -1 of the fungus DNA, with repeatability. Real time PCR presented higher sensitivity, having the detecting limit determined for the technique with repeatability, at the concentration of 10 fg of DNA of the fungus. In two farms 24 external asymptomatic leaves from orange trees variety Pera Rio were collected; eight from each third (lower, medium and upper); totaling twent plants. The modified CTAB method for DNA extraction was used. The presence of the fungus in very low concentrations was detected in the asymptomatic leaves, which made it impossible when we used the conventional PCR technique. In those conditions, the real-time PCR proved to be feasible, reproducible and highly sensitive for G. citricarpa detection, amplifying between 232 and 232 x 10 2 DNA copies of the fungus from asymptomatic leaf samples; being an excellent option for the diagnosis of thispathogen in asymptomatic orchards.
A pinta preta dos citros (PPC), causada pelo fungo Guignardia citricarpa, é considerada uma doença quarentenária, que impõe restrições ao transporte de frutas frescas para países da União Europeia. Em Goiás, apesar dos sistemáticos levantamentos fitossanitários, ainda não se conhece a real distribuição da ocorrência da PPC em função da tecnologia disponível para o diagnóstico. A ocorrência de infecção latente, a presença de uma espécie endofítica muito semelhante morfologicamente à G. citricarpae o tempo para o diagnóstico por métodos convencionais levam à necessidade de validar métodos moleculares rápidos, eficientes, reprodutíveis, seguros e sensíveis de diagnóstico, que garantem confiabilidade dos diagnósticos e dos levantamentos de detecção e delimitação da distribuição dessa doença. Foram testadas modificações do método “Cationic hexadecyl trimethyl ammonium bromide” (CTAB) para extração de DNA de G. citricarpaem tecidos de frutos cítricos sintomáticos, com a avaliação dapureza química, integridade estrutural e ausência de substâncias inibidoras na solução de DNA, para posterior uso em diagnóstico por reação em cadeia da polimerase (PCR). Não houve diferença significativa na quantidade média de DNA extraído para tecidos higienizados e não higienizados (328,62 ng µL -1 e 322,79 ng µL -1 , respectivamente), com baixa concentração de proteínas na suspensão de DNA. A obtenção de DNA em quantidade (>300 ng µL -1 ) e qualidade (integridade estrutural) foi suficiente para realização das análises de PCR convencional e PCR em tempo real. A ausência de compostos inibidores foi demostrada por PCR em tempo real pela adição de DNA padrão de milho, geneticamente modificado 0,1 % (evento MON 810 padrão ERM®-BF413b), com a amplificação da região específica desse evento em todas as amostras teste. O método CTAB modificado apresentou repetitividade e reprodutibilidade parcial dentro dos limites aceitáveis para a metodologia, apresentando coeficientes de variação inferiores a 30 %. Em atendimento à norma ISO/IEC 17025:2005, foi validado o método para diagnóstico do fungo G. citricarpa por PCR convencional e PCR em tempo real com a avaliação da especificidadee do limite de detecção. Os métodos de PCR convencional e PCR em tempo real demonstraram serem específicos e adequados para a detecção de G. citricarpa. A PCR convencional apresentou o limite de detecção de 10 ng µL -1 de DNA do fungo, com repetitividade. A PCR em tempo real apresentou maior sensibilidade, sendo o limite de detecção determinado para a técnica, com repetitividade, na concentração 10 fg de DNA do fungo. Em duas propriedades foram coletadas 24 folhas externas assintomáticas de laranja-pera rio, sendo oito em cada terço (inferior, médio e superior) da planta, em um total de vinte plantas. Para a extração do DNA, foi utilizado o método CTAB modificado. Em folhas assintomáticas, apresença do fungo foi detectada em baixíssimas concentrações, o que inviabiliza a utilização da técnica PCR convencional. Nessas condições, a PCR em tempo real demonstrou ser viável, reprodutível e altamente sensível para a detecção de G. citricarpa, sendo detectado na concentração de 232 a 232 x 10² números de cópia do DNA do fungo em amostras de folhas assintomáticas, constituindo uma excelente opção para o diagnóstico desse patógeno em pomares assintomáticos.
Silveira, Ismênia Glauce de Oliveira Barreto da. "Perfil clínico-epidemiológico, sorológico e molecular da hanseníase em área endêmica potiguar." Universidade Federal Rural do Semi-Árido, 2017. http://bdtd.ufersa.edu.br:80/tede/handle/tede/701.
Full textMade available in DSpace on 2017-05-19T20:57:26Z (GMT). No. of bitstreams: 1 Ismênia_DISSERT.pdf: 2286785 bytes, checksum: fe512f9cb09ab8ccaeca35cce30794cc (MD5) Previous issue date: 2017-02-17
Leprosy is a chronic infectious disease caused by Mycobacterium leprae and persists as a serious public health problem in Brazil. The fact that this microorganism is inculcable makes it difficult to diagnose and elucidate details of its transmissive chain. Aiming to clarify the role of house dust in the disease transmission route, this case-control study carried out in the Barrocas, Mossoró, RN, Brazil, the largest cluster in the state, compared clinical, epidemiological, slit skin smear and serological results of 22 newly diagnosed patients of the cluster, with molecular results of detection of the specific RLEP and 16S rRNA genomic regions of this bacillus in the nasal swab samples, saliva and house dust of these individuals and of the controls (44 household contacts and 44 endemic contacts) between november 2015 to november 2016. There was greater detection in women and school-age youth, with degree of disability, with predominance of paucibacillary forms. There was no statistical difference between the number of BCG vaccine scars from patients and endemic contacts, and 31.8% of the patients had no vaccine scar. The two rapid serological tests evaluated, ML-flow and OrangeLife® presented similar results, with a higher positivity among paucibacillary patients through the latter (54.5%). Both were superior to slit skin smear (9.1%). Regarding molecular research, the positivity in nasal swab and saliva of multibacillary patients with primer RLEP was 16.7% and 33.3%, respectively. There was no detection of bacterial DNA in domestic dust or between paucibacillary. Regarding the DNA analysis in saliva of endemic multibacillary contacts, there was 27.2% positivity. Associating molecular and serological results, it was possible to identify 4.5% of subclinicity between home contacts and 15% of asymptomatic carrier status. Regarding the risk variables, residing in dwellings with up to two windows offered 3.79 times more chance of progressing to this outcome, having a history of cases of leprosy in the family increased 2.89 times the risk of presenting seropositivity by the OrangeLife® serological test In this community and being over 60 years of age gave 3.6 times more chances of having positive serological reaction for leprosy with the test evaluated, which denotes prolonged exposure to the bacillus. Although not statistically significant, it was noted that the pattern of low schooling, low income, environmental insalubrity, population agglomeration and inadequate living habits made up a sociodemographic and epidemiological profile that attests to the context of social vulnerability of the affected population, requiring integrated actions of rehabilitation that go beyond the physical aspect of their dwellings, since they need to contemplate the human and social development of these people in the broad sense
A hanseníase é uma doença infecciosa crônica causada pelo Mycobacterium leprae e persiste como grave problema de saúde pública no Brasil. O fato deste micro-organismo ser incultivável dificulta o diagnóstico e elucidação de detalhes da sua cadeia transmissiva. Objetivando analisar a dinâmica de transmissão ambiental da hanseníase, este estudo caso-controle realizado no Bairro Barrocas, Mossoró/RN, maior cluster do estado, confrontou resultados de avaliação clínica, epidemiológica, baciloscópica e sorológica de 22 doentes recém-diagnosticados nas Barrocas, com resultados moleculares de detecção das regiões genômicas específicas RLEP e 16S rRNA deste bacilo nas amostras de swab nasal, saliva e poeira domiciliar destes indivíduos e dos seus controles (44 contactantes domiciliares e 44 contactantes endêmicos), entre novembro de 2015 a novembro de 2016. Os indivíduos foram diagnosticados através de busca ativa nos domicílios, nas escolas e na sede da unidade básica de saúde local. Houve maior detecção da doença em mulheres, nos menores de 15 anos, com grau 0 de incapacidade e predominância de formas paucibacilares. Não houve diferença estatística entre o número de cicatrizes vacinais de BCG de doentes e de contatos endêmicos, e 31,8% dos hansenianos não apresentaram cicatriz vacinal. Os dois testes rápidos sorológicos avaliados, ML-flow (IgM ND-O-BSA) e OrangeLife® (IgM e IgG anti NDO-LID 1) apresentaram resultados semelhantes, com maior positividade entre os paucibacilares através deste último (54,5%). Ambos foram superiores à baciloscopia (9,1%). Quanto à pesquisa molecular, a positividade em swab nasal e saliva de doentes multibacilares com primer RLEP foi de 16,7% e 33,3%, respectivamente. Não houve detecção de DNA bacteriano na poeira doméstica nem entre os paucibacilares. Já em relação à pesquisa de DNA em saliva de contatos endêmicos de multibacilares, houve 27,2% de positividade. Associando-se resultados moleculares e sorológicos, foi possível identificar 4,5% de subclinicidade e 15% de status portador assintomático entre contatos domiciliares. Em relação às variáveis de risco de soropositivar com o teste OrangeLife®, este estudo revelou que residir em moradias com até duas janelas ofereceu 3,79 vezes mais chance de evoluir para este desfecho, ter histórico de casos de hanseníase na família aumentou 2,89 vezes o risco e ter mais de 60 anos de idade conferiu 3,6 vezes mais chances, o que denota exposição prolongada destes indivíduos ao bacilo. Embora sem significância estatística, fez-se notório que o padrão de baixa escolaridade, baixa renda, insalubridade ambiental, aglomeração populacional e hábitos de vida inadequados compuseram um perfil sociodemográfico e epidemiológico que atestou o contexto de vulnerabilidade social da população afetada, exigindo ações integradas de reabilitação que vão além do aspecto físico de suas moradias, pois necessitam contemplar o desenvolvimento humano e social dessas pessoas no seu sentido amplo
2017-05-18
Bispo, Paulo José Martins [UNIFESP]. "Aplicação de métodos moleculares no diagnóstico de endoftalmite bacteriana." Universidade Federal de São Paulo (UNIFESP), 2009. http://repositorio.unifesp.br/handle/11600/8956.
Full textObjetivo: 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
Chandramoulee, Swaran Yuvaneswari. "Evaluation of direct PCR for forensic DNA profiling and the development of a direct PCR multiplex." Thesis, University of Strathclyde, 2012. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=18935.
Full textGarnier, Fabien. "Identification au niveau de l'espèce de streptocoques du groupe viridans par PCR." Paris 5, 1996. http://www.theses.fr/1996PA05P164.
Full textSantos, Mónica Cristina Barão Costa. "PCT e PCR no apoio ao diagnóstico de sépsis no recém-nascido." Master's thesis, Universidade de Aveiro, 2012. http://hdl.handle.net/10773/10891.
Full textApesar de uma significativa diminuição nas últimas décadas, os processos infeciosos mantêm-se como uma das principais causas de mortalidade/morbilidade neonatal, um fato em parte devido à imaturidade do sistema imunológico do recém-nascido (RN) e ao crescente número de natos prematuros de reduzido peso corporal. O diagnóstico precoce de quadros de sépsis e a apropriada intervenção terapêutica representam deste modo um desafio diário em neonatologia. Dos diversos marcadores biológicos utilizados no apoio ao diagnóstico de sépsis, as proteínas de fase aguda procalcitonina (PCT) e proteína C-reactiva (PCR) têm mostrado considerável utilidade clínica. Devido à sua rápida cinética após estímulo imunológico, são considerados marcadores biológicos precoces de sépsis, permitindo uma resposta em tempo útil inferior ao tempo necessário para a identificação do agente etiológico por exame cultural. Pretendeu-se com este trabalho reavaliar a utilidade dos marcadores biológicos PCT e PCR no apoio ao diagnóstico de sépsis no RN. Adicionalmente foi também avaliada a possível correlação entre o padrão de variação dos referidos biomarcadores e a etiologia dos agentes infeciosos. O estudo baseou-se na recolha de dados relativos aos parâmetros PCT e PCR em RN com suspeita de sépsis, seguindo-se o seu tratamento e análise estatística. As amostras foram processadas no Centro Hospitalar Entre Douro e Vouga (CHEDV) dizendo respeito ao intervalo de tempo compreendido entre Janeiro de 2011 e Abril de 2012. Os resultados obtidos corroboram com outros estudos feitos para o mesmo efeito, em que a PCT e a PCR em conjugação com uma cuidadosa avaliação clínica, fornecem uma informação de grande valia no diagnóstico de sépsis no RN. Relativamente aos resultados dos exames culturais pedidos nas primeiras horas de vida dos 84 RN do estudo, 99% foram negativos e 1% foram positivos. Estes resultados sustentam crescentes dúvidas acerca da utilidade/validade dos exames culturais.
In the last recent decades, the infectious processes have been decreasing significantly; however, they are still one of the leading causes of neonatal mortality / morbidity, in part due to the immaturity of the newborn immune system and to the increasing number of premature births, whose infants are of low birth weight. Early diagnosis of sepsis and the appropriate therapeutic intervention represent, therefore, a daily challenge in neonatology units. Among all the different biological markers used to support the diagnosis of sepsis, acute phase proteins, procalcitonin (PCT) and C-reactive protein (CRP) have shown to be of considerable clinical value. Due to their rapid kinetics after immune stimulation, these molecules are considered early markers of sepsis, as they provide the needed answers earlier than the time usually needed for the identification of the etiologic agents done by culture exam. The aim of this study was to reassess the utility of the biomarkers PCT and CRP in order to help in the diagnosis of newborn sepsis. Additionally, it was also evaluated the possible correlation between the variation profile of these biomarkers and the etiology of the infectious agents. The study was based on the data collection of the PCT and CRP parameters in neonates, who were under sepsis suspicion, followed by the treatment and statistical analysis. The samples were processed in the Centro Hospitalar Entre Douro e Vouga (CHEDV), which comprise the analysed cases from January 2011 till April 2012. The results corroborate other studies done for the same effect, in which the PCT and CRP in conjunction with a careful clinical assessment, provide valuable information in the diagnosis of sepsis in neonates. In what culture test results ordered in the first 84 hours of life of infants in the study is concerned, 99% were negative and 1% was positive. These results support the growing doubts about the usefulness / validity of the cultural tests.
Dutra, Mauricio Cabral. "Perfil de eliminação de agentes infecciosos envolvidos em rinite na espécie suína." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-22042009-102538/.
Full textRespiratory diseases are one of the largest cause of economic losses in swine industry, it is related with grown and weight gain reduction, mortality, vaccines and medicaments costs, veterinary assistance. In that context, rinithis cases have been a major contribution. The present study propose the determination of elimination profile of agents related with rhinitis evaluating different ages in nine swine herds with history of cornet lesions and that uses different ways to control and prevent this problem. There were examined tonsils swabs from 12 animals in the following ages: sows, piglets of 20, 40 60 days and pigs of 90, 110 and 140 days, totalizing 84 pigs for farm. The swabs were searched to P. multocida capsular type A and D, dermonecrotic toxin gene from P. multocida, B. bronchiseptica and porcine cytomegalovirus through polymerase chain reaction (PCR). Despite de turbinate bones lesions present in all herds P. multocida type A was detected in only one pig and none were positive to dermonecrotic toxin gene. From 756 animals, 22 (2.9%) were positive to B. bronchiseptica and 198 (26.1%) to porcine cytomegalovirus detection. The presence of B. bronchiseptica presented statistical association with the farrowing and finishing times. Larger number of animals positive to cytomegalovirus show statistical association with the post weaning pigs. Sows carrying B. bronchiseptica in the three types of herds examined, suggesting that sows have an active participation in piglet infection by this agent. The same was not observed in porcine cytomegalovirus spread. More projects were need to clarify the low detection of P. multocida and to understand the impact of cytomegalovirus in swine production.
Remualdo, Vanessa Rosália. "Amplificação do DNA de Mycobacterium tuberculosis presente em amostras de esfregaço bucal, pela técnica de reação em cadeia da polimerase (PCR)." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/23/23141/tde-17062009-170152/.
Full textMycobacterium tuberculosis is the causing agent of the tuberculosis, responsible illness for 26% of the prevention deaths in the entire world. In Brazil 85000new cases are notified annually, being esteem 50 million people contaminated by the M. tuberculosis. It is considered priority disease for the control of illnesses for the Health department. For this control, it has to be reliable methods and resources for the ready laboratorial diagnosis. Bacterioscopiv, histological analysis or culture of the microrganism from samples of sputum are techniques normally used. The limitation of these methods is low sensitivity and long-winded 8 weeks. The PCR is one technique of amplification of DNA, that if has shown promising instrument for the diagnosis of the tuberculosis. The M. tuberculosis is a microorganism that has affinity for the cells (intracellular microorganism) and can be present in the cells of the respiratory treat and the oral mucosa. Oral swab, in contrast of sputum, is easily taken, not invasive and offering lesser risks of contamination for other microorganisms. We analyze 80 samples of oral swab of patients with confirmed diagnosis of tuberculosis, of these, 78 (97,4%) had resulted positive in the PCR. We conclude that the oral swab use and the application of the PCR are an effective and trustworthy method for tuberculosis detention of the M. tuberculosis.
Castilla, Karina Salvagni. "Detecção de genes de virulência em diferentes fagotipos e ribotipos de Salmonella Enteritidis utilizando a Reação em Cadeia da Polimerase (PCR)." Universidade de São Paulo, 2003. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-13052004-145302/.
Full textThis study has the intention of verify the four virulence genes event in Salmonella Enteritidis according with phage type and ribotype and the virulence in vivo. The genes studied were invA, spvC, sefC and pefA in 120 Salmonella Enteritidis isolates from different origins, belongs at different ribotypes and phage types of seven Brazilian states. To verify the genes presence the strains were examined by Polimerase Chain Reaction (PCR) technique, singly and multiplex. The event of invA gene was in 100% (120/120) of strains, the spvC gene was in 94% (113/120) of strains, the sefC gene was in 97.5% (117/120) of strains and the pefA gene was in 97% (116/120) of strains. There were discovered five different profiles. The pattern one, P1, was positive for invA, spvC, sefC e pefA. The P2 was positive for invA, spvC and pefA. The P3 was positive for invA, sefC e pefA. The P4 was positive for invA and sefC and the last one, P5, was positive for invA and spvC. For the PT4RT1 strains, the P1 profile was present in 94% (64/68) of strains; P2 profile was in 1.5% (1/68); P3 in 3% (2/68); and P4 in 1.5% (1/68) of strains. For the PT4RT2 strains, the P1 profile was present in 86% (18/21) of strains; P3 profile was in 5% (1/21); and P4 in 9% (2/21) of strains. For the PT4RT3 strains, the P1 profile was present in 80% (4/5) of strains, and P2 in 20% (1/5) of strains. For the PT4RT9 strains, the P1 profile was present in 91% (10/11) of strains, and P3 in 9% (1/11) of strains. The strains: PT4RT5, PT7RT1 e PT4RT10 belongs at the P1 profile, and only the PT1 strain belongs at the P5s profile. The virulence was evaluated challenging the birds orally and subcutaneous, through the colonization of the caecum, liver and spleen invasion. The gene spvC was related with the survivance and the increase of the average grow of the bacteria in the liver and spleen, but this study doesnt demonstrate the difference in the percentage of positive birds for the bacteria in their organs. The sefC and pefA genes were important to promote the caecum colonization, because only when both genes were present simultaneously in the profile, obtain the caecum isolation when the birds were been by orally challenged this way the main route of the infection of this pathogen.
Bischoff, Erik. "Schnellnachweis von bierschädlichen Bakterien mit PCR." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=964420333.
Full textLien, Tonje Gulbrandsen. "Statistical Analysis of Quantitative PCR Data." Thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for matematiske fag, 2011. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-13094.
Full textKarlsson, Magdalena, and Emilia Semberg. "Tracing probiotics in salami using PCR." Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-157177.
Full textHalpern, Micah. "Immuno-PCR detection of Lyme borreliosis." Doctoral diss., University of Central Florida, 2013. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/6286.
Full textPh.D.
Doctorate
Molecular Biology and Microbiology
Medicine
Biomedical Sciences
Feugeas, Olivier. "Pcr (polymerase chain reaction) et vih." Lille 2, 1990. http://www.theses.fr/1990LIL2M264.
Full textPelikánová, Veronika. "Automatická genotypizace bakterií metodou rep-PCR." Master's thesis, Vysoké učení technické v Brně. Fakulta elektrotechniky a komunikačních technologií, 2018. http://www.nusl.cz/ntk/nusl-378029.
Full textŠuranská, Hana. "Identifikace vinných kvasinek metodou PCR-RFLP." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2009. http://www.nusl.cz/ntk/nusl-216494.
Full textReis, Levi Eduardo Soares. "Detecção de Leishmania por PCR e suas variações (seminested PCR e PCR em tempo real), em fragmentos de pele e de baço de cães com leishmaniose visceral." Programa de Pós-Graduação em Ciências Farmacêuticas. CIPHARMA, Escola de Farmácia, Universidade Federal de Ouro Preto, 2013. http://www.repositorio.ufop.br/handle/123456789/3364.
Full textApproved for entry into archive by Gracilene Carvalho (gracilene@sisbin.ufop.br) on 2013-10-21T14:45:17Z (GMT) No. of bitstreams: 2 license_rdf: 23599 bytes, checksum: 9e2b7f6edbd693264102b96ece20428a (MD5) DISSERTAÇÃO_DetecçãoLeishmaniaPCR.pdf: 1009268 bytes, checksum: f3643332a0889fcb5d973bed29de784f (MD5)
Made available in DSpace on 2013-10-21T14:46:00Z (GMT). No. of bitstreams: 2 license_rdf: 23599 bytes, checksum: 9e2b7f6edbd693264102b96ece20428a (MD5) DISSERTAÇÃO_DetecçãoLeishmaniaPCR.pdf: 1009268 bytes, checksum: f3643332a0889fcb5d973bed29de784f (MD5) Previous issue date: 2013
A detecção do DNA de Leishmania spp, pela Reação em Cadeia da Polimerase (PCR) e suas variações, surgem como alternativas para o diagnóstico da LVC, por serem métodos altamente sensíveis e específicos. O objetivo deste trabalho foi comparar a PCR e suas variações (Seminested PCR e PCR em tempo real) utilizando amostras de pele e de baço de 60 cães soropositivos (RIFI e ELISA). Os animais foram agrupados considerando a forma clínica, sendo classificados como assintomáticos (CA; n=20), oligossintomáticos (CO; n= 22) e sintomáticos (CS; n= 18). Como controle negativo, foram utilizados fragmentos de três cães não infectados provenientes do canil da UFOP. O diagnóstico parasitológico, utilizado como padrão ouro, foi realizado por meio de duas metodologias: cultivo de aspirado medular em meio de cultura NNN/LIT e pela visualização direta de formas amastigotas do parasito em lâminas com imprints de pele e de baço. As análises moleculares foram realizadas utilizando iniciadores direcionados para a região conservada do minicírculo do kDNA de Leishmania – L150/L152 e LINR4/LIN17/LIN19 para as técnicas de PCRc e snPCR, respectivamente. Na qPCR foram utilizados iniciadores que amplificam o gene da DNA polimerase (DNA pol α) de L. infantum. De acordo com os testes parasitológicos 61,7% das amostras foram positivas. Nos fragmentos de pele, a sensibilidade da PCRc foi de 89,2%, já para a snPCR e qPCR foi de 86,5% e 97,3% respectivamente. VPP para a PCRc foi de 36,0%, snPCR de 35,3% e da qPCR foi 38,1%. O VPN foi de 93,6%, 92,1% e 98,3% pelas técnicas de PCRc, snPCR e qPCR respectivamente. Em amostras de baço, a sensibilidade da PCRc foi 81,1%, snPCR de 94,6% e de 100,0% pela qPCR. O VPP para a PCRc foi 33,9%, snPCR 37,4% e qPCR 38,7%. O VPN da PCRc foi de 89,3%, snPCR 96,7% e qPCR 100,0%. A positividade nos testes moleculares aumentou de acordo com a gravidade dos sinais clínicos. Foi observado que a qPCR apresentou os melhores resultados na pele e no baço devido a maior sensibilidade, VPP e VPN, em comparação as outras técnicas moleculares. Sendo assim, concluímos que a melhor técnica e tecido para o diagnóstico molecular da LVC é a qPCR de pele, devido à elevada sensibilidade e fácil obtenção da amostra biológica. ____________________________________________________________________________________
ABSTRACT: The detection of Leishmania DNA based on polymerase chain reaction (PCR) and its variations represent alternatives for CVL diagnosis with highly sensitive and specific methods. The aim of this work was to compare the PCR and its variations (Seminested PCR and quantitative PCR) in samples of skin and spleen of 60 seropositive dogs (IFAT and ELISA). The animals were divided according to their clinical presentation and were classified as asymptomatic (CA, n = 20), oligosymptomatic (CO, n = 22) and symptomatic (CS, n = 18). As a negative control, we used fragments of three uninfected dogs from the kennel of UFOP. The parasitological diagnosis used as gold standard was performed by two methods: culture of bone marrow aspirate in culture medium NNN/LIT and direct visualization of amastigotes of the parasite on slides with imprints of skin and spleen. The primers L150/L152 and LINR4/LIN17/LIN19 were used to amplify the conserved region of the Leishmania kDNA minicircle in the cPCR and snPCR and qPCR were performed out using the DNA polymerase gene (DNA pol α) primers from L. infantum. According to the parasitological test 61.7% the samples were positive. In skin samples, the sensitivity of cPCR was 89.2%, snPCR was 86.5% and qPCR was 97.3%. PPV for cPCR was 36.0%, snPCR was 35.3% and qPCR was 38.1%. The NPV was 93.6%, 92.1% and 98.3% by the techniques of cPCR, snPCR and qPCR. In spleen samples, the sensitivity of cPCR was 81.1%, snPCR was 94.6% and qPCR was 100.0%. PPV for cPCR was 33.9%, snPCR was 37.4% and qPCR was 38.7%. NPV for cPCR was 89.3%, snPCR was 96.7% and qPCR was 100.0%. Positivity in molecular tests increased with the progression of clinical signs. It was observed that the qPCR showed the best results in skin and spleen due to higher sensitivity, PPV and NPV compared to other molecular techniques. Thus, we conclude that the best technique and tissue for molecular diagnosis of CVL is the qPCR skin due to the high sensitivity and easy obtaining the biological sample.
Palmer, Ruth Helen. "Cloning and characterisation of the PRK family : a novel family of protein kinases related to the PKC superfamily." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321771.
Full textSolinas, Antonio. "Novel approaches in genetic analysis." Thesis, University of Southampton, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274529.
Full textKribii, Rachida. "La squalène synthase d'Arabidopsis thaliana - caractérisation moléculaire et expression chez la levure Saccharomyces cerevisiae." Poitiers, 1996. http://www.theses.fr/1996POIT2390.
Full textBANERJEE, SHOMIR. "A PROTOTYPE ON-CHIP MICRO-HEATER FOR DISPOSABLE MICRO-PCR MODULE." University of Cincinnati / OhioLINK, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1038001719.
Full textMoreno, Ana Cláudia Almeida. "Diagnóstico molecular na era da sequenciação de 3ª geração e da PCR digital." Master's thesis, [s.n.], 2013. http://hdl.handle.net/10284/3969.
Full textO campo científico do diagnóstico molecular vive atualmente tempos áureos de desenvolvimento. A busca de tecnologias revolucionárias capazes de fornecer informação de um modo rápido, preciso e a baixo custo, nunca foi tão intensa. A descoberta do DNA e a necessidade urgente do seu estudo, impulsionaram o desenvolvimento de novas técnicas de análise, como a reação em cadeia da polimerase (PCR) e a sequenciação. Desde então, estes procedimentos não pararam de evoluir, constituindo hoje um pilar essencial para a investigação e prática clínica. A PCR, uma técnica de biologia molecular capaz de amplificar sequencias especificas de moléculas de DNA, é atualmente um método indispensável e rotineiro na investigação médica e cientifica. Desde a primeira técnica de PCR à PCR digital, investigadores em todo o mundo procuraram desenvolver ferramentas cada vez mais poderosas e eficazes, capazes de serem utilizadas em todos os campos científicos. A demanda por um método que permitisse desvendar a informação codificada no genoma humano impulsionou a evolução das técnicas de sequenciação. A sequenciação pelo método dideoxy, desenvolvido por Sanger, foi a base para a atual sequenciação de terceira geração. O aparecimento destas novas técnicas proporcionou uma inexorável evolução tecnológica marcada por um elevado rendimento num curto espaço de tempo. Análises genómicas que anteriormente eram luxos inalcançáveis, tornaram-se progressivamente mais acessíveis pela diminuição significativa do custo por análise. No futuro próximo, os diagnósticos moleculares continuarão a desempenhar um papel critico na qualidade da saúde da população mundial. Inúmeras técnicas de testes moleculares permitem a deteção e caracterização das mais variadas doenças, assim como a monitorização da sua terapêutica. É importante perceber como cada uma destas técnicas funciona e qual a mais indicada para cada tipo de laboratório. Neste trabalho, procuramos oferecer ao leitor uma visão alargada das vantagens, limitações e aplicações associadas a cada tecnologia atualmente disponível. These are exciting times for scientists in the field of molecular diagnostics. The demand for revolutionary technologies that deliver a fast, inexpensive and accurate genomic information has never been greater. This journey began with the discovery of DNA. The urge to study this vital molecule led to the development of new and revolutionizing techniques, such as the polymerase chain reaction (PCR) and sequencing. Since then, these procedures never stopped evolving and nowadays provide vital information for clinical practice. The PCR is a scientific technique in molecular biology that is able to amplify a single piece of a particular DNA sequence. It is now a common and often indispensable method used in medical and biological research labs for a variety of applications. Between the first PCR and the digital PCR available nowadays, researchers sought to develop more effective and powerful tools to be used in all scientific fields. The quest to find a method able to unveil the information behind the human genome pushed the DNA sequencing tools to the next level. The dideoxy terminator sequencing developed by Sanger was the workhorse to develop what we currently call the 3rd generation sequencing. The arrival of these new next generation techniques caused an enormous technological shift marked by dramatic throughput increases, in a less time consuming process. Genomic analysis that were, for most, unreachable luxuries just a few years ago, are being increasingly democratized at a rapid pace due to striking decreases in the cost of each run. In the upcoming years, molecular diagnostics will continue to be of critical importance to worldwide public health. There are innumerous techniques available for molecularbased tests for detection and characterization of disease, as well as monitoring of drug response. It is important to understand how each of these techniques work and which one would be more suitable in each particular laboratory. In this paper we seek to give the reader a good grasp of the advantages and limitations associated to the technological machinery available today.
Reck, Carolina. "Detecção de Mycoplasma synoviae e Orthoreovirus aviario em lesões de artrites em frangos e matrizes de corte." Universidade do Estado de Santa Catarina, 2011. http://tede.udesc.br/handle/handle/840.
Full textCoordenação de Aperfeiçoamento de Pessoal de Nível Superior
Infectious arthritis in broiler and breeders represents an economic and health problem resulting in severe losses due to retarded growth and down grading at slaughterhouse . The most commom agents associated with cases of infectious arthritis in avian are Mycoplasma synoviae (MS) and Orthoreovirus avian (ARV). The objective of this study was to evaluate the occurence of MS and ARV in infectious arthritis through molecular techniques, such as PCR (Polymerase Chain Reaction) and RT-PCR (Reverse Transcription - Polymerase Chain Reaction). In addition, we carried out histopathological studies of joints with arthritis and development of a multiplex PCR. For the study 150 samples were collected from lesions of broilers and 180 samples of breeders. MS were detected in 82.6% and ARV 20% of the simple analysis in breeders. The analysis of samples from broilers showed positivity to MS (58.9%) and ARV (33.9%). The histopathology analysis of joints demonstrated the presence of infiltrate of heterophils in the synovial capsule and the digital flexor tendon. The inflammatory process has also been found in samples from joints that were negative by PCR for both agents.The developed multiplex, with satisfactory results, amplifying genes from MS and ARV simultaneamente. In conclusions that molecular techniques, PCR and PCR-M, were effective for detecting MS and ARV in lesions of arthritis from breeders and broiler
A artrite infecciosa em frangos e matrizes de corte representa um problema sanitário e econômico de grande impacto, provocando elevadas perdas de produtividade e nos processos de produção e industrialização. O impacto econômico relacionado às perdas por problemas no aparelho locomotor normalmente são subestimados, pois geralmente são consideradas apenas as perdas no abatedouro, não contabilizando os descartes que ocorrem durante o período de alojamento.Os principais agentes etiológicos associados aos casos de artrites infecciosas em aves são o Mycoplasma synoviae (MS) e o Orthoreovirus aviario (ARV). O presente trabalho teve por objetivo detectar a presença de MS e ARV através de técnicas moleculares, PCR (Polymerase Chain Reaction) e RT-PCR (Reverse Transcription - Polymerase Chain Reaction), respectivamente, em casos de artrites em frangos e matrizes de corte. Além disso, realizou-se estudos histopatológicos das articulações com artrites e desenvolvimento de um multiplex PCR. Para o estudo foram coletadas 150 amostras de lesões artritícas de matrizes de corte e 180 amostras de frango de corte, durante o período de 2009 e 2010. Do total das 150 amostras de matriz de corte submetidas a PCR , sendo que 82,6% (124/150) foram positivas para MS e 20%(30/150) foram positivas para ARV. Em 83,3% (25/30) foi possível detectar a presença de ARV e MS na mesma articulação . A análise das amostras de frango de corte demonstrou positividade para o MS em 58,9% (106/180) das amostras e 33,9% (61/180) foram positivas para o ARV. A análise histopatológica das lesões demonstrou a presença de processo inflamatório crônico na cápsula sinovial e no tendão flexor digital. O processo inflamatório foi encontrado também nas amostras de articulações que foram negativas na PCR para ambos os agentes. A multiplex desenvolvida, apresentou resultados satisfatórios, amplificando genes do MS e ARV simultaneamente. Conclui-se que as técnicas moleculares, PCR, RT-PCR e M-PCR, mostraram-se eficientes para detecção de MS e ARV em lesões de artrite de matrizes e frango de corte, podendo ser incorporadas na rotina de diagnóstico para os respectivos agentes a partir de material articular
Noll, Lance. "Escherichia coli O157: detection and quantification in cattle feces by quantitative PCR, conventional PCR, and culture methods." Thesis, Kansas State University, 2015. http://hdl.handle.net/2097/18923.
Full textDepartment of Diagnostic Medicine/Pathobiology
T. G. Nagaraja
Shiga toxin-producing E. coli O157 is a major foodborne pathogen. The organism colonizes the hindgut of cattle and is shed in the feces, which serves as a source of contamination of food. Generally, cattle shed E. coli O157 at low concentrations (≤ 10[superscript]2 CFU/g), but a subset of cattle, known as “super-shedders”, shed high concentrations (>10[superscript]3 CFU/g) and are responsible for increased transmission between animals and subsequent hide and carcass contamination. Therefore, concentration data are an important component of quantitative microbial risk assessment. A four-plex quantitative PCR (mqPCR) targeting rfbE[subscript]O157, stx1, stx2 and eae was developed and validated to detect and quantify E. coli O157 in cattle feces. Additionally, the applicability of the assay to detect E. coli O157 was compared to conventional PCR (cPCR) targeting the same four genes, and a culture method. Specificity of the assay to differentially detect the four genes was confirmed. In cattle feces spiked with pure cultures, detection limits were 2.8 x 10[superscript]4 and 2.8 x 10[superscript]0 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. Of the 100 samples that were randomly picked from the 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from the 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar’s chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. Applicability of the assay to quantify E. coli O157 was determined with feedlot cattle fecal samples (n=576) and compared to spiral plate method. Fecal samples that were quantifiable for O157 by mqPCR (62/576; 10.8%) were at concentrations of ≥ 10[superscript]4 CFU/g of feces. Only 4.5% (26/576) of samples were positive by spiral plate method, with the majority (17/26; 65.4%) at below 10[superscript]3 CFU/g. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect and quantify E. coli O157 in cattle feces.
Lima, Ana Carolina Stocco de. "Diagnóstico molecular de Leishmaniose Tegumentar Americana: identificação de espécies de Leishmania por SSUrDNA PCR e G6PD PCR." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/5/5160/tde-20092010-162811/.
Full textAmerican Cutaneous Leishmaniasis (ACL) presents a serious problem of public healthy. In Brazil many species are recognized as pathogenic to humans, therefore differential diagnostic is necessary to understand the epidemiological profile of ACL in endemic areas. Fifty-three paraffinembedded skin biopsies of ACL patients from Pará (N=33) and Maranhão (20) States, were submitted to different protocols of polymerase chain reaction (PCR) for identification of their causative agents. Biopsies were deparaffinized and DNA were extracted using phenol-chloroform, quantified and submitted to PCR reaction, using small subunit coding sequence (SSUrDNA) and enzyme glucose-6-phosphate dehydrogenase (G6PD). The target of G6PD was used both in conventional PCR reactions (cPCR) and quantitative PCR (qPCR). The reactions of cPCR and Nested PCR SSUrDNA showed positive result for the genus Leishmania in 40 (83.3%) of the samples. Twenty-seven positive samples were submitted to sequencing and 10 were identified as L. (Viannia) sp. and 17 as L. (L.) amazonensis. The G6PD PCR identified 9 samples as L. (V.) braziliensis, 2 from Pará (6%) and 7 from Maranhão (35%).Four samples were quantified in G6PD qPCR, even this is a single copy. These results indicate that specific sequences from Leishmania sp. present in multiple copies should be chosen in relation to those from unique copies in paraffin-embedded tissues, once is frequent cases of ACL with low parasitism, consequently small DNA concentrations and that SSUrDNA can be a good target to diagnostic and epidemiologic studies of ACL. The qPCR allowed the detection and identification of L. (Viannia) sp. in a single round of amplification in four samples that when showed positive results only in the Nested or Semi Nested cPCR suggesting a higher sensitivity offered by qPCR