Avalia????o de m??todos de extra????o de DNA e de identifica????o de dermat??fitos por an??lise de PCR-RFLP

• Author(s): Frota, Maria Zeli Moreira
• Date: 2017
• Origin: Oasisbr
• Subject(s): Dermat??fitos; Extra????o de DNA; PCR-RFLP; Topoisomerase II; CI??NCIAS BIOL??GICAS
• Document Type: Doctoral thesis
• Language: Portuguese

Description

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Dermatophytes comprise a group of filamentous fungi of great interest on public health because of their ability to parasitize keratinized tissues, such as skin, hair and nails, and for their wide distribution in the world. As a consequence of this parasitism, an infectious process of dermatophytosis is established, from which a variety of clinical manifestations can occur, affecting people of both genders and all age groups. Laboratory methods for mycological diagnosis do not always allow a clear an especific definition of the agent. In this study, different strategies for extraction of DNA, and molecular typing by PCR-RFLP, of seven dermatophyte species were assessed. Two target regions: ITS/rDNA and the topoisomerase II gene were evaluated, by testing three PCR protocols and three restriction enzymes (DdeI, HinfI, HaeIII). For the DNA extraction, the glass bead shaking technique for cell lysis, followed by Gustincich (1991) based mehod for DNA separation, demonstrated more advantages. Our results has demonstrated that the topoisomerase II gene is a suitable target region for identification of the seven major pathogenic dermatophyte fungal species, reinforcing previous studies, and pointed to a new PCR-RFLP protocol, which is based on a PCR of this gene using dPsD2 primer, followed by digestion of PCR products with HaeIII restriction enzyme.

Os dermat??fitos compreendem um grupo de fungos filamentosos de grande interesse na ??rea da sa??de, devido ?? sua capacidade de parasitar os tecidos queratinizados, como a pele, p??los e unhas, e ?? sua ampla distribui????o no mundo. Como conseq????ncia desse parasitismo instala-se um processo infeccioso de dermatofitose, a partir do qual pode ocorrer uma diversidade de manifesta????es cl??nicas, acometendo pessoas de ambos os g??neros e de todos os grupos et??rios. Os m??todos laboratoriais para o diagn??stico micol??gico nem sempre permitem uma clara defini????o do agente em n??vel de esp??cie. No presente estudo foram analisadas diferentes estrat??gias para a extra????o de DNA e para a identifica????o molecular por PCR-RFLP das principais esp??cies de dermat??fitos. Duas regi??es alvo, a regi??o ITS/DNAr e o gene da topoisomerase II foram analisadas, testando-se tr??s protocolos de PCR e tr??s enzimas de restri????o (DdeI, HinfI, HaeIII). Na extra????o do DNA, o m??todo de lise utilizando p??rolas de vidro e a separa????o do DNA com base no m??todo de Gustincich (1991) demonstrou importantes vantagens. Nossos resultados demonstraram que o gene da topoisomerase II ?? uma regi??o alvo adequada para identifica????o das sete principais esp??cies de fungos dermat??fitos patog??nicos, refor??ando estudos anteriores, e apontaram para um novo protocolo de RFLP-PCR, que se baseia em uma PCR desse gene utilizando o primer dPsD2, seguido da digest??o dos produtos obtidos com a enzima de restri????o HaeIII.