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Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / O gênero Enterococcus inclui aproximadamente 30 espécies, algumas das quais muito freqüentemente envolvidas com alimentos e ambientes relacionados, e cuja diferenciação com identificação precisa torna-se muitas vezes problemático, quando estas questões baseiam-se apenas em características fenotípicas que, mesmo assim, permitem separar estes microrganismos em cinco grupos fisiologicamente diferentes. A dificuldade para categorizar inequivocamente as espécies dos enterococos com base somente nas características bioquímicas pode estar relacionado com uma heterogeneidade bastante alta dos aspectos fenotípicos, independentemente da origem dos isolados, devendo ainda ser considerado o fato de haver bastante similaridade das exigências nutricionais entre os enterococos e outras bactérias ácido lácticas. Por estas razões as técnicas moleculares tornaram-se um importante instrumento para caracterização destes microrganismos. Dentre estas, aquelas que se baseiam no uso do 16S rRNA como molécula alvo têm sido consideradas como excelentes opções com propósitos de identificação. A técnica PCR/RFLP fundamenta-se em uma metodologia baseada na amplificação por PCR e nos perfis de restrição obtidos após o uso de enzimas selecionadas com base na sua habilidade de revelar polimorfismo nos fragmentos de DNA ou RNA analisados. Este trabalho tem como objetivo implantar a técnica da PCR/RFLP com as enzimas DdeI, HaeIII e HinfI associada a alguns testes bioquímicos para uma identificação de Enterococcus spp. isolados de amostras de queijo Minas tipo frescal. / The genus Enterococcus includes about 30 species, some of them very often involved with food and related environments, and whose differentiation with precise identification often becomes problematic when based only on phenotypic characteristics. Anyway this techniques allow to separate these microorganisms in five groups physiologically different. Enterococci are often difficult to clearly categorize in species based only on these phenotypic characteristics. In addition to having a very high diversity of phenotypic aspects, regardless of the origin of the isolates, it must to be considered that there are enough similarities between the nutritional requirements of enterococci and other lactic acid bacteria. For these reasons the molecular techniques have become important tools for characterizing these microorganisms. Among these, those that are based on the use of 16S rRNA as a target molecule have been considered as excellent options for any identification. PCR/RFLP is a method based on the PCR amplification and on the restriction profiles obtained by using restriction enzymes selected based on their ability to reveal polymorphism in fragments of DNA or RNA analysed. This work aims to introduce the technique of PCR/RFLP with the enzymes DdeI, HaeIII and HinfI associated with certain biochemical tests for identification of Enterococcus spp. isolated from samples of fresh Minas cheese. Twenty-one reference strains of the genus Enterococcus were used to establish the restriction profiles. Fifty-four isolates from milk and chicken that had phenotypic profiles typical, atypical and without some identification, stocked in our laboratory, were used to test the technique developed, and 21 isolates from the fresh Minas cheese were used to confirm the usefulness of the technique. By using the PCR/RFLP we obtained 5 different restriction profiles and, when associated with some biochemical tests, it was possible to obtain a rapid and accurate identification of most species of Enterococcus.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.arca.fiocruz.br:icict/4012 |
| Date | January 2009 |
| Creators | Scheidegger, Érica Miranda Damasio |
| Contributors | Leite, Paola Cardarelli |
| Source Sets | IBICT Brazilian ETDs |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Source | reponame:Repositório Institucional da FIOCRUZ, instname:Fundação Oswaldo Cruz, instacron:FIOCRUZ |
| Rights | info:eu-repo/semantics/openAccess |
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