Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae)

Varela, João Bosco

Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae)

Detalhes bibliográficos
Autor(a) principal:	Varela, João Bosco
Data de Publicação:	2016
Tipo de documento:	Dissertação
Idioma:	por
Título da fonte:	Biblioteca Digital de Teses e Dissertações da UFRRJ
Texto Completo:	https://rima.ufrrj.br/jspui/handle/20.500.14407/11915
Resumo:	O estudo de carrapatos e doenças transmitidas por eles é cada vez mais dependente da utilização de técnicas de biologia molecular que são empregadas na detecção de patógenos, e a acurada identificação desses artrópodes, em particular os estágios imaturos. A aplicação bem-sucedida dos métodos moleculares, principalmente, a reação em cadeia da polimerase (PCR), só pode ser alcançada se o DNA presente no carrapato foi eficazmente preservado e extraído de forma eficiente. O estudo comparou três fixadores (RNAlater, sais de zinco (ZN) e isopropanol) avaliando a sua capacidade de preservar DNA mitocondrial (mtDNA) e nuclear de larvas e ninfas de Amblyomma parvum e larvas de Amblyomma scultptum. O DNA foi extraído dos carrapatos, em tempos entre 72h e 12 meses após a fixação por meio da técnica de fenol-clorofórmio e lise alcalina (“Hot Shot”) e examinadas usando ensaios de PCR para sequências nucleares (espaçador interno transcrito 2; ITS2) e mitocondriais (12S rDNA, subunidade 1 do citocromo c oxidase (COI) e D-loop). A eficiência de amplificação foi analisada quantitativamente (número de amostras que produziram “amplicon”) e qualitativamente (intensidade relativa das bandas observadas em géis de agarose). Foi observado que a sequência ITS2 foi amplificada na maioria (93,39%, n= 283/303) das amostras em cada um dos três fixadores, embora tenham sido observadas diferenças qualitativas, particularmente com A. sculptum preservado em ZN. Em contraste, a fixação em isopropanol afetou negativamente a capacidade de amplificar as sequências dos marcadores mitocondriais de A. parvum e também resultou na amplificação inferior (qualitativa), do alvo D-loop com A. sculptum. Esses efeitos foram observados em amostras fixadas por apenas 72 horas. Os efeitos prejudiciais de isopropanol também foram observados igualmente em amostras extraídas usando um método de lise alcalina (“HotShot”). As amostras de larvas de A. parvum preservadas em RNAlater durante 30 meses, mostrou uma eficácia de amplificação de 100% nos ensaios para ITS2 e COI, independentemente do método de extração usado. Sequências mitocondriais são um componente central da maioria das pesquisas moleculares com carrapatos. Os resultados deste estudo indicam que isopropanol deve ser evitado como um fixador para fases imaturas de carrapatos. Em vez disso, o uso de RNAlater é recomendado, a fim de permitir a recuperação consistente de mtDNA amplificáve

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spelling	Varela, João BoscoMcIntosh, Douglas5404662719http://lattes.cnpq.br/5166697605343047Santos, Huarrisson AzevedoOgrzewalska, María Halina426.722.588-56http://lattes.cnpq.br/86551772397073592023-12-22T01:58:41Z2023-12-22T01:58:41Z2016-02-24VARELA, João Bosco. Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae). 2016. 60 f. Dissertação (Mestrado em CIÊNCIAS VETERINÁRIAS). Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, 2016.https://rima.ufrrj.br/jspui/handle/20.500.14407/11915O estudo de carrapatos e doenças transmitidas por eles é cada vez mais dependente da utilização de técnicas de biologia molecular que são empregadas na detecção de patógenos, e a acurada identificação desses artrópodes, em particular os estágios imaturos. A aplicação bem-sucedida dos métodos moleculares, principalmente, a reação em cadeia da polimerase (PCR), só pode ser alcançada se o DNA presente no carrapato foi eficazmente preservado e extraído de forma eficiente. O estudo comparou três fixadores (RNAlater, sais de zinco (ZN) e isopropanol) avaliando a sua capacidade de preservar DNA mitocondrial (mtDNA) e nuclear de larvas e ninfas de Amblyomma parvum e larvas de Amblyomma scultptum. O DNA foi extraído dos carrapatos, em tempos entre 72h e 12 meses após a fixação por meio da técnica de fenol-clorofórmio e lise alcalina (“Hot Shot”) e examinadas usando ensaios de PCR para sequências nucleares (espaçador interno transcrito 2; ITS2) e mitocondriais (12S rDNA, subunidade 1 do citocromo c oxidase (COI) e D-loop). A eficiência de amplificação foi analisada quantitativamente (número de amostras que produziram “amplicon”) e qualitativamente (intensidade relativa das bandas observadas em géis de agarose). Foi observado que a sequência ITS2 foi amplificada na maioria (93,39%, n= 283/303) das amostras em cada um dos três fixadores, embora tenham sido observadas diferenças qualitativas, particularmente com A. sculptum preservado em ZN. Em contraste, a fixação em isopropanol afetou negativamente a capacidade de amplificar as sequências dos marcadores mitocondriais de A. parvum e também resultou na amplificação inferior (qualitativa), do alvo D-loop com A. sculptum. Esses efeitos foram observados em amostras fixadas por apenas 72 horas. Os efeitos prejudiciais de isopropanol também foram observados igualmente em amostras extraídas usando um método de lise alcalina (“HotShot”). As amostras de larvas de A. parvum preservadas em RNAlater durante 30 meses, mostrou uma eficácia de amplificação de 100% nos ensaios para ITS2 e COI, independentemente do método de extração usado. Sequências mitocondriais são um componente central da maioria das pesquisas moleculares com carrapatos. Os resultados deste estudo indicam que isopropanol deve ser evitado como um fixador para fases imaturas de carrapatos. Em vez disso, o uso de RNAlater é recomendado, a fim de permitir a recuperação consistente de mtDNA amplificáveThe study of ticks and tick-borne disease is increasingly dependent upon the use of molecular biological techniques that are employed in pathogen detection and for the accurate identification of ticks, particularly immature stages. The successful application of molecular methods, principally the polymerase chain reaction (PCR), can only be achieved if the DNA present in the tick was effectively preserved and could be extracted efficiently. The current study compared three fixatives (RNAlater, zinc salts (ZN) and isopropanol) for the ability to preserve the nuclear and mitochondrial (mt) DNA of larvae and nymphs of Amblyomma parvum and larvae of Amblyomma scultptum. DNA was extracted from ticks at times between 72h to 12 months post fixation using a phenol-chloroform procedure and examined using PCR assays for nuclear (internal transcribed spacer 2; ITS2) and mitochondrial (12S rDNA, subunit 1 of cytochrome c oxidase (COI) and D-loop) sequences. The efficiency of amplification was analyzed quantitatively (number of samples which produced amplicon) and qualitatively (relative intensity of bands observed on agarose gels). It was observed that the ITS2 sequence could be amplified in the majority (93,39%, n= 283/303) of the samples, in each of the three fixatives, although qualitative differences were observed, particularly with A. sculptum preserved in ZN. In contrast, fixation in isopropanol effectively abolished the ability to amplify the mitochondrial marker sequences of A. parvum and also resulted in inferior amplification (qualitative), of the D-loop target with A. sculptum. Those effects were observed in samples fixed for as little as 72h. The detrimental effects of isopropanol were also observed in samples extracted using an alkaline lysis method (Hotshot). Samples of A. parvum larvae preserved in RNAlater for 30 months showed an amplification efficacy of 100% in the ITS2 and COI assays, irrespective of the extraction method. Mitochondrial sequences are a central component of the majority of molecular studies of ticks. The findings of this study indicate that isopropanol should be avoided as a fixative for immature stages of ticks. Instead, the use of RNAlater is recommended in order to permit the consistent recovery of amplifiable mtDNAapplication/pdfporUniversidade Federal Rural do Rio de JaneiroPrograma de Pós-Graduação em Ciências VeterináriasUFRRJBrasilInstituto de VeterináriaDNA mitocondrialAmblyomma parvumAmblyomma sculptummitochondrial DNAnuclear DNApreservationDNA nuclearpreservaçãoPCRMedicina VeterináriaInfluência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae)Influence of conservation methods upon retrieval and amplification fequency of mitochondrial and nuclear markers from Amblyomma parvum and Amblyomma sculptum ticks (Acari: Ixodidae)info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisAGUIAR, D. M. et al. 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dc.title.por.fl_str_mv	Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae)
dc.title.alternative.eng.fl_str_mv	Influence of conservation methods upon retrieval and amplification fequency of mitochondrial and nuclear markers from Amblyomma parvum and Amblyomma sculptum ticks (Acari: Ixodidae)
title	Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae)
spellingShingle	Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae) Varela, João Bosco DNA mitocondrial Amblyomma parvum Amblyomma sculptum mitochondrial DNA nuclear DNA preservation DNA nuclear preservação PCR Medicina Veterinária
title_short	Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae)
title_full	Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae)
title_fullStr	Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae)
title_full_unstemmed	Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae)
title_sort	Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae)
author	Varela, João Bosco
author_facet	Varela, João Bosco
author_role	author
dc.contributor.author.fl_str_mv	Varela, João Bosco
dc.contributor.advisor1.fl_str_mv	McIntosh, Douglas
dc.contributor.advisor1ID.fl_str_mv	5404662719
dc.contributor.advisor1Lattes.fl_str_mv	http://lattes.cnpq.br/5166697605343047
dc.contributor.referee1.fl_str_mv	Santos, Huarrisson Azevedo
dc.contributor.referee2.fl_str_mv	Ogrzewalska, María Halina
dc.contributor.authorID.fl_str_mv	426.722.588-56
dc.contributor.authorLattes.fl_str_mv	http://lattes.cnpq.br/8655177239707359
contributor_str_mv	McIntosh, Douglas Santos, Huarrisson Azevedo Ogrzewalska, María Halina
dc.subject.eng.fl_str_mv	DNA mitocondrial Amblyomma parvum Amblyomma sculptum mitochondrial DNA nuclear DNA preservation
topic	DNA mitocondrial Amblyomma parvum Amblyomma sculptum mitochondrial DNA nuclear DNA preservation DNA nuclear preservação PCR Medicina Veterinária
dc.subject.por.fl_str_mv	DNA nuclear preservação PCR
dc.subject.cnpq.fl_str_mv	Medicina Veterinária
description	O estudo de carrapatos e doenças transmitidas por eles é cada vez mais dependente da utilização de técnicas de biologia molecular que são empregadas na detecção de patógenos, e a acurada identificação desses artrópodes, em particular os estágios imaturos. A aplicação bem-sucedida dos métodos moleculares, principalmente, a reação em cadeia da polimerase (PCR), só pode ser alcançada se o DNA presente no carrapato foi eficazmente preservado e extraído de forma eficiente. O estudo comparou três fixadores (RNAlater, sais de zinco (ZN) e isopropanol) avaliando a sua capacidade de preservar DNA mitocondrial (mtDNA) e nuclear de larvas e ninfas de Amblyomma parvum e larvas de Amblyomma scultptum. O DNA foi extraído dos carrapatos, em tempos entre 72h e 12 meses após a fixação por meio da técnica de fenol-clorofórmio e lise alcalina (“Hot Shot”) e examinadas usando ensaios de PCR para sequências nucleares (espaçador interno transcrito 2; ITS2) e mitocondriais (12S rDNA, subunidade 1 do citocromo c oxidase (COI) e D-loop). A eficiência de amplificação foi analisada quantitativamente (número de amostras que produziram “amplicon”) e qualitativamente (intensidade relativa das bandas observadas em géis de agarose). Foi observado que a sequência ITS2 foi amplificada na maioria (93,39%, n= 283/303) das amostras em cada um dos três fixadores, embora tenham sido observadas diferenças qualitativas, particularmente com A. sculptum preservado em ZN. Em contraste, a fixação em isopropanol afetou negativamente a capacidade de amplificar as sequências dos marcadores mitocondriais de A. parvum e também resultou na amplificação inferior (qualitativa), do alvo D-loop com A. sculptum. Esses efeitos foram observados em amostras fixadas por apenas 72 horas. Os efeitos prejudiciais de isopropanol também foram observados igualmente em amostras extraídas usando um método de lise alcalina (“HotShot”). As amostras de larvas de A. parvum preservadas em RNAlater durante 30 meses, mostrou uma eficácia de amplificação de 100% nos ensaios para ITS2 e COI, independentemente do método de extração usado. Sequências mitocondriais são um componente central da maioria das pesquisas moleculares com carrapatos. Os resultados deste estudo indicam que isopropanol deve ser evitado como um fixador para fases imaturas de carrapatos. Em vez disso, o uso de RNAlater é recomendado, a fim de permitir a recuperação consistente de mtDNA amplificáve
publishDate	2016
dc.date.issued.fl_str_mv	2016-02-24
dc.date.accessioned.fl_str_mv	2023-12-22T01:58:41Z
dc.date.available.fl_str_mv	2023-12-22T01:58:41Z
dc.type.status.fl_str_mv	info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv	info:eu-repo/semantics/masterThesis
format	masterThesis
status_str	publishedVersion
dc.identifier.citation.fl_str_mv	VARELA, João Bosco. Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae). 2016. 60 f. Dissertação (Mestrado em CIÊNCIAS VETERINÁRIAS). Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, 2016.
dc.identifier.uri.fl_str_mv	https://rima.ufrrj.br/jspui/handle/20.500.14407/11915
identifier_str_mv	VARELA, João Bosco. Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae). 2016. 60 f. Dissertação (Mestrado em CIÊNCIAS VETERINÁRIAS). Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, 2016.
url	https://rima.ufrrj.br/jspui/handle/20.500.14407/11915
dc.language.iso.fl_str_mv	por
language	por
dc.relation.references.por.fl_str_mv	AGUIAR, D. M. et al. Prevalence of Ehrlichia canis (Rickettsiales: Anaplasmataceae) in dogs and Rhipicephalus sanguineus (Acari: Ixodidae) ticks from Brazil.Journal of medical entomology, v. 44, n.1, p. 126-132, 2007. ALVES, A.D.S. et al. Seroprevalence of Rickettsia spp. in Equids and Molecular Detection of ‘Candidatus Rickettsia amblyommii’in Amblyomma cajennense Sensu Lato Ticks From the Pantanal Region of Mato Grosso, Brazil. Journal of Medical Entomology, v. 51, n. 6, p. 1242-1247, 2014. AMMAZZALORSO, A.D. et al. To beat or not to beat a tick: Comparison of DNA extraction methods from ticks (Ixodes scapularis) (No. e1042). PeerJ PrePrints, p. 1-17, 2015. ANDERSON, John F.; MAGNARELLI, Louis A. Biology of Ticks. Infectious Disease Clinics of North America, v. 22, n. 2, p. 195–215, 2008. DE ARMAS, Y.; RODRÍGUEZ, M.M.; BISSET, J.A., Modificacion de un método de extracción de ADN genómico de Aedes aegypti (Diptera: Culicidae). 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Influência dos métodos de conservação sobre a recuperação e a frequência de amplificação de marcadores mitocondriais e nuclueares de carrapatos das espécies Amblyomma Parvum e Amblyomma Sculptum (Acari: Ixodidae)

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