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DC Field | Value | Language |
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dc.contributor.author | Machado, Lucas Aguiar Rosa | |
dc.date.accessioned | 2023-12-22T01:57:34Z | - |
dc.date.available | 2023-12-22T01:57:34Z | - |
dc.date.issued | 2022-02-16 | |
dc.identifier.citation | MACHADO, Lucas Aguiar Rosa. Expansão e aprimoramento de ferramenta on-line baseada no uso da técnica de PCR-RFLP para identificação de espécies do gênero Amblyomma Koch, 1844 (Acari: Ixodidae). 2022. 82 f. Dissertação (Mestrado em Ciências Veterinárias) - Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Seropédica, 2022. | por |
dc.identifier.uri | https://rima.ufrrj.br/jspui/handle/20.500.14407/11827 | - |
dc.description.abstract | Durante os últimos 20 anos, pesquisadores brasileiros desempenharam um papel de liderança nos esforços para melhorar o nosso conhecimento da bioecologia, presença de patógenos e dinâmica das populações de carrapatos associados com animais silvestres na América do Sul. Muito destes achados foram fornecidos na forma de dados moleculares, principalmente sequenciamento de marcadores moleculares amplificados usando a técnica da “polymerase chain reaction” (PCR). Pesquisas elaboradas no PPGCV/UFRRJ por nosso grupo, entre 2018-2019, resultaram no desenvolvimento de um sistema robusto e de baixo custo (que serve como uma alternativa ao sequenciamento) para a identificação, em nível de espécie, de carrapatos da família Ixodidae. O sistema é baseado na técnica de PCR-RFLP (do inglês “Polymerase Chain Reaction-Restriction Fragment Length Polymorphism”), utilizando um fragmento do gene mitocondrial que codifica o RNA ribossômico 16S como alvo. Para facilitar o acesso a esse sistema e promover seu uso por outros pesquisadores brasileiros, foi desenvolvida uma ferramenta “on-line” denominada “TickCutter”. O presente projeto visou expandir e aperfeiçoar esta ferramenta em duas frentes. Em primeiro lugar, uma nova ferramenta denominada “COIsearcher” foi desenvolvida usando um marcador molecular alternativo, especificamente Citocromo C Oxidase I (COI), com o intuito de resolver algumas das limitações associadas com o uso de um único marcador molecular. Em segundo lugar através da inclusão de dados de PCR-RFLP do gene 16S rDNA com as enzimas DraI e VspI, oriundos de espécies de carrapatos da família Argasidae ou provenientes de novos representantes da família Ixodidae. Um total de 35 novos padrões de bandas foram identificados entre 363 sequências inéditas de 16S rDNA procedentes de carrapatos da família Ixodidae. Em relação aos carrapatos da família Argasidae, foram identificados 31 perfis de bandas entre as 75 sequências obtidas do GenBank (os quais representaram 22 espécies de Argasídeos). No entanto, sete padrões procedentes de seis espécies de Argasidae geraram identificações conflitantes com perfis derivados de alguns carrapatos da família Ixodidae. A nova ferramenta “COIsearcher” foi desenvolvida de modo bastante semelhante a ferramenta “TickCutter 16S” e aproveitou de algumas das soluções elaboradas para a resolução de problemas encontrados durante o desenvolvimento do módulo 16S. No entanto, algumas modificações foram necessárias devido as diferenças entre os marcadores, particularmente em termos do tamanho dos “amplicons”. O banco de dados do novo módulo foi estabelecido com base na digestão in silico de “amplicons” virtuais (709 nucleotídeos) do gene COI com as enzimas AluI e MboI, oriundos de espécies de carrapatos da família Ixodidae. Um total de 88 perfis de bandas foram registrados entre as 851 sequências procedentes de 33 das 51 espécies da família Ixodidae. Foram detectadas algumas identificações conflitantes com a ferramenta “COIsearcher”, de modo similar ao notado anteriormente com a ferramenta “TickCutter 16S”. A solução encontrada para resolver estes conflitos foi a identificação de uma terceira enzima capaz de gerar padrões discriminatórios em nível de espécies. As modificações introduzidas na plataforma “TickCutter” fornecerão maior flexibilidade e poder discriminatório ao sistema de identificação atual e oferecerá uma solução para a maioria das limitações associadas ao uso de um único marcador molecular. | por |
dc.description.sponsorship | CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior | por |
dc.description.sponsorship | CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico | por |
dc.description.sponsorship | FAPERJ - Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro | por |
dc.format | application/pdf | * |
dc.language | por | por |
dc.publisher | Universidade Federal Rural do Rio de Janeiro | por |
dc.rights | Acesso Aberto | por |
dc.subject | Acarologia | por |
dc.subject | enzimas de restrição | por |
dc.subject | biologia molecular | por |
dc.subject | Acarology | eng |
dc.subject | Restriction enzymes | eng |
dc.subject | Molecular biology | eng |
dc.title | Expansão e aprimoramento de ferramenta on-line baseada no uso da técnica de PCR-RFLP para identificação de espécies do gênero Amblyomma Koch, 1844 (Acari: Ixodidae) | por |
dc.title.alternative | Expansion and Improvement of an On-line Tool Based on the Use of PCR-RFLP for Identification of Species Within the Genus Amblyomma Koch, 1844 (Acari: Ixodidae) | eng |
dc.type | Dissertação | por |
dc.description.abstractOther | During the last 20 years, Brazilian researchers have played a leading role in efforts to improve our knowledge of the bioecology, the presence and importance of pathogens and the dynamics of tick populations associated with wild animals in South America. Many of these findings were provided in the form of molecular data, principally sequencing of molecular markers amplified using the polymerase chain reaction (PCR) technique. Research carried out by our group within the PPGCV/UFRRJ, between 2018-2019, resulted in the development of a robust and low-cost system (which serves as an alternative to sequencing) for the identification, at the species level, of ticks of the Ixodidae family. The system is based on the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism technique (PCR-RFLP), using a fragment of the mitochondrial gene that encodes 16S ribosomal RNA as target. To facilitate access to this system and to promote its use by other Brazilian researchers, an online tool called “TickCutter” was developed. The present project aimed to expand and improve this tool on two fronts. Firstly, a new tool called “COIsearcher” was developed using an alternative molecular marker, specifically Cytochrome C Oxidase I (COI), in order to address some of the limitations associated with the use of a single molecular marker. Secondly, through the inclusion of PCR-RFLP data of the 16S rDNA gene with the enzymes DraI and VspI, derived from tick species of the Argasidae family and/or from novel representatives of the Ixodidae family. A total of 35 new banding patterns were identified among 363 previously unpublished 16S rDNA sequences derived from ticks of the Ixodidae family. Regarding the Argasidae family of ticks, 31 banding profiles were identified among the 75 sequences obtained from GenBank (which represented 22 Argasidae). However, it was observed that patterns derived from six species of Argasidae generated conflicting identifications with banding profiles derived from certain species of the Ixodidae family. The new “COIsearcher” tool was developed in a very similar manner to the “TickCutter 16S” tool, and took advantage of some of the solutions developed to solve problems encountered during the development of the 16S module. However, some modifications were necessary due to the differences between the markers, particularly in terms of the size of the amplicons. The database of the new module was established based on the in silico digestion of virtual “amplicons” (709 nucleotides) of the COI gene, derived from tick species of the Ixodidae family, with the enzymes AluI and MboI. A total of 88 banding profiles were recorded among a total of 851 sequences derived from 33 of the 51 species of the Ixodidae family. The interspecific discriminatory power of the “COIsearcher” tool was high. However, some conflicting identifications were detected, in a manner similar to what was previously observed with the “TickCutter 16S” tool. The solution found to resolve these conflicts was the identification of a third enzyme capable of generating discriminatory patterns at the species level. It was concluded that the modifications introduced to the “TickCutter” platform will provide greater flexibility and discriminatory power to the current identification system and will offer a solution to most of the limitations associated with the use of a single molecular marker. | eng |
dc.contributor.advisor1 | McIntosh, Douglas | |
dc.contributor.advisor1ID | 054.046.627-19 | por |
dc.contributor.referee1 | McIntosh, Douglas | |
dc.contributor.referee2 | Luz, Hermes Ribeiro | |
dc.contributor.referee3 | Abreu, Daniel Paiva Barros de | |
dc.creator.ID | 134.395.857-19 | por |
dc.creator.Lattes | http://lattes.cnpq.br/7955108206654178 | por |
dc.publisher.country | Brasil | por |
dc.publisher.department | Instituto de Veterinária | por |
dc.publisher.initials | UFRRJ | por |
dc.publisher.program | Programa de Pós-Graduação em Ciências Veterinárias | por |
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dc.subject.cnpq | Medicina Veterinária | por |
dc.subject.cnpq | Parasitologia | por |
dc.thumbnail.url | https://tede.ufrrj.br/retrieve/69242/2022%20-%20Lucas%20Aguiar%20Rosa%20Machado.pdf.jpg | * |
dc.originais.uri | https://tede.ufrrj.br/jspui/handle/jspui/5637 | |
dc.originais.provenance | Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2022-05-10T19:10:28Z No. of bitstreams: 1 2022 - Lucas Aguiar Rosa Machado.pdf: 2507813 bytes, checksum: 35960d966bbfa67c05e9c96e41d723e7 (MD5) | eng |
dc.originais.provenance | Made available in DSpace on 2022-05-10T19:10:28Z (GMT). No. of bitstreams: 1 2022 - Lucas Aguiar Rosa Machado.pdf: 2507813 bytes, checksum: 35960d966bbfa67c05e9c96e41d723e7 (MD5) Previous issue date: 2022-02-16 | eng |
Appears in Collections: | Mestrado em Ciências Veterinárias |
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Files in This Item:
File | Description | Size | Format | |
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2022 - Lucas Aguiar Rosa Machado.pdf | 2.45 MB | Adobe PDF | ![]() View/Open |
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