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Previous issue date: 2016-02-24 / The 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 mtDNA / O estudo de carrapatos e doen?as transmitidas por eles ? cada vez mais dependente da utiliza??o de t?cnicas de biologia molecular que s?o empregadas na detec??o de pat?genos, e a acurada identifica??o desses artr?podes, em particular os est?gios imaturos. A aplica??o bem-sucedida dos m?todos moleculares, principalmente, a rea??o em cadeia da polimerase (PCR), s? pode ser alcan?ada se o DNA presente no carrapato foi eficazmente preservado e extra?do de forma eficiente. O estudo comparou tr?s 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 extra?do dos carrapatos, em tempos entre 72h e 12 meses ap?s a fixa??o por meio da t?cnica de fenol-clorof?rmio e lise alcalina (?Hot Shot?) e examinadas usando ensaios de PCR para sequ?ncias nucleares (espa?ador interno transcrito 2; ITS2) e mitocondriais (12S rDNA, subunidade 1 do citocromo c oxidase (COI) e D-loop). A efici?ncia de amplifica??o foi analisada quantitativamente (n?mero de amostras que produziram ?amplicon?) e qualitativamente (intensidade relativa das bandas observadas em g?is de agarose). Foi observado que a sequ?ncia ITS2 foi amplificada na maioria (93,39%, n= 283/303) das amostras em cada um dos tr?s fixadores, embora tenham sido observadas diferen?as qualitativas, particularmente com A. sculptum preservado em ZN. Em contraste, a fixa??o em isopropanol afetou negativamente a capacidade de amplificar as sequ?ncias dos marcadores mitocondriais de A. parvum e tamb?m resultou na amplifica??o 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 tamb?m foram observados igualmente em amostras extra?das usando um m?todo de lise alcalina (?HotShot?). As amostras de larvas de A. parvum preservadas em RNAlater durante 30 meses, mostrou uma efic?cia de amplifica??o de 100% nos ensaios para ITS2 e COI, independentemente do m?todo de extra??o usado. Sequ?ncias mitocondriais s?o 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 ? recomendado, a fim de permitir a recupera??o consistente de mtDNA amplific?ve
| Identifer | oai:union.ndltd.org:IBICT/oai:localhost:jspui/1365 |
| Date | 24 February 2016 |
| Creators | Varela, Jo?o Bosco |
| Contributors | McIntosh, Douglas, Santos, Huarrisson Azevedo, Ogrzewalska, Mar?a Halina |
| Publisher | Universidade Federal Rural do Rio de Janeiro, Programa de P?s-Gradua??o em Ci?ncias Veterin?rias, UFRRJ, Brasil, Instituto de Veterin?ria |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ |
| Rights | info:eu-repo/semantics/openAccess |
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