Validação de técnicas moleculares para o diagnóstico de bactérias em peixes, visando redução de tempo e custo

• Ano de defesa: 2015
• Autor(a) principal: Sebastião, Fernanda de Alexandre [UNESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Estadual Paulista (Unesp)
• Palavras-chave em Português: Peixes; Bacterias; Biologia molecular; Reação em cadeia de polimerase; Seqüenciamento de nucleotídeo; Genes; Fishes
• Link de acesso: http://hdl.handle.net/11449/123993

With the intensification of the Brazilian fish farming, characterized by increasing density storage, especially in cages, it has been observed a growing number of diseases, mainly bacterial, causing high mortality and significant economic losses in all phases of breedin. One of the problems related to the control of diseases in fish farming is the constant misuse of antibiotics and chemotherapeutics that cause problems of bacterial resistance and environmental impacts which affect the quality of the final product. Thus, the search for rapid and effective diagnostic alternative is increasingly necessary, since they contribute to control of diseases before they cause irreversible clinical signs and high mortality rates. In view of this, the molecular diagnostic techniques represented by the Polymerase Chain Reaction (PCR) and its variations, such as sequencing and real time PCR, constitute alternatives to be explored. Thus, the objectives of this work were: a) adapting the methodology of colony PCR coupled with sequencing of the 16S rRNA gene for bacteria that cause disease in fish, b) determine the sensitivity to antimicrobial florfenicol and oxytetracycline bacterial genera of higher frequency, c) validate real-time PCR assay for detection and quantification of Aeromonas hydrophila and Streptococcus agalactiae and to evaluate its sensitivity and analytical specificity d) applying the validated Methodology for the diagnosis of infections caused by these pathogens in infected experimentally tilapias groups. In the developed methodologies, we attempted to reduce costs and time to diagnosis. The results showed that: colony PCR showed a reduction of costs and time compared to traditional isolation techniques, biochemical identification, DNA extraction and conventional PCR; the alliance of colony PCR and 16S rRNA gene sequencing allowed identification of all tested bacterial pathogens, not being necessary the design of specific ...