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Detection of Influenza A Viruses From Environmental Lake and Pond IceKoçer, Zeynep A. 09 July 2010 (has links)
No description available.
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Thermodynamics and Kinetics of Subunit Assembly in HIV-1 Reverse TranscriptaseVenezia, Carl Frank 23 January 2010 (has links)
No description available.
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Studies of HIV-1 mutagenesis during drug therapy and the molecular determinants of HIV-1 variationChen, Renxiang 29 September 2004 (has links)
No description available.
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Real-time RT-PCR analysis of two epitope regions encoded by the VP2 gene of infectious bursal disease virusesMickael, Claudia Silva 14 July 2005 (has links)
No description available.
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Quantitative analysis of individual flue-cured tobacco seed tissues reveals Tobacco mosaic virus infection in embryosEllis, Madeleine D. 28 June 2019 (has links)
Tobacco mosaic virus (TMV) is an extensively studied RNA virus that reduces quality and yield in commercially grown tobacco (Nicotiana tabacum L.). The virus is transmitted mechanically, although infections have been associated with contaminated seeds with the seed coat being the source of virus. Thus, TMV transmission is said to be seedborne (as opposed to true seed transmission where the embryo is infected). The objective of this study was to identify TMV concentrations in the three components of an individual tobacco seed: seed coat (SC), endosperm (ED), and embryo (EM). Six hundred seed from TMV infected K 326 flue-cured cultivar tobacco plants were carefully dissected into the three components. Total RNA was extracted from each sample and synthesized into cDNA for analysis. A quantitative real-time PCR (RT-qPCR) assay was developed to quantify viral titers in each component, while endpoint PCR confirmed RT- qPCR results and established a threshold viral cycle (Ct) value. Endpoint PCR results revealed viral accumulation in all three components of a tobacco seed. The highest concentration of TMV was in the SC, followed by ED and EM. A similar viral concentration gradient was observed in each individual tobacco seed from all three experimental plants. This is the first detection of TMV in tobacco embryos and suggests the virus can be seed transmitted. / Master of Science / Tobacco mosaic virus (TMV) is a widely studied plant virus that affects tobacco, tomato, pepper, and many other crops throughout the world. The virus is easily transmitted through contaminated tools or machinery, workers’ hands or clothing, or when an infected leaf comes into contact with an uninfected leaf. For years, TMV transmission was said to be seedborne, not seed transmitted, meaning that seedling infection comes from the infected external seed coat of the seed. Seed transmission of the virus has yet to be proven because of the difficulty to fully separate tobacco seed tissues. The objective of this study was to identify TMV concentrations in the three components of an individual tobacco seed: seed coat (SC), endosperm (ED), and embryo (EM). Six hundred seed from TMV infected tobacco plants were carefully dissected into the three components. Total RNA was extracted from each sample, and synthesized into cDNA for analysis. A quantitative real-time polymerase chain reaction (RT-qPCR) assay was developed to quantify viral concentrations in each component. Endpoint PCR was used to confirm the quantitative results of RT-qPCR. Results revealed TMV accumulation in all three components of a tobacco seed, the highest concentration detected in the SC, followed by ED and EM; this pattern was observed from each plant. This is the first report of TMV being detected in embryos of tobacco seed which suggests that TMV can be seed transmitted.
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Characterization, toxicity, and biological activities of organometallic compounds and peptide nucleic acids for potential use as antimicrobialsErnst, Marigold Ellen Bethany 29 April 2019 (has links)
Bacterial antibiotic resistance is a globally recognized problem that has prompted extensive research into novel antimicrobial compounds. This dissertation describes research focusing on two types of potential antimicrobial molecules, organometallic compounds (OMC) and peptide nucleic acids (PNA). Organometallic compounds show promise as antimicrobial drugs because of their structural difference from conventional antibiotics and antimicrobials, and because of the ability to "tune" their chemical and biological properties by varying ligand attachments. Peptide nucleic acids, when linked to a cell-penetrating peptide (CPP), can suppress bacterial gene expression by an antisense mechanism and are attractive candidates for antimicrobial drugs because they bind strongly to target nucleic acids and are resistant to nucleases. Chapters 1 and 2 of the dissertation provide an introduction and broad literature review to frame the experimental questions addressed. Chapter 3 describes work to test the cytotoxicity and cellular penetration capabilities of novel OMCs by evaluating their effects on J774A.1 murine macrophage-like cells that were either uninfected or were infected with Mycobacterium bovis BCG. Results indicate that OMCs with an iridium (Ir) metal center and an amino acid ligand show minimal cytotoxicity against eukaryotic cells but likely do not penetrate the intracellular compartment in significant amounts. Chapter 4 presents research into in vitro effects of CPP-PNAs targeting the tetA and tetR antibiotic resistance genes (CPP-anti-tetA PNA and CPP-anti-tetR PNA, respectively) in tetracycline-resistant Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Through the use of modified minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays it was shown that both the CPP-anti-tetA PNA and CPP-anti-tetR PNA increase tetracycline susceptibility in DT104. Chapter 5 explores the molecular mechanism of the CPP-anti-tetA PNA and CPP-anti-tetR PNA through the use of reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results indicate good specificity of the CPP-anti-tetA PNA for its nucleic acid target as evidenced by suppression of tetA mRNA expression in DT104 cultures treated with a combination of tetracycline and the PNA. Chapter 6 describes the development of a mouse model of DT104 infection using BALB/c mice, followed by implementation of that model to test in vivo antimicrobial effects of the CPP-anti-tetA PNA and the CPP-Sal-tsf PNA, which targets expression of the essential tsf gene. An optimal dose of DT104 was identified that causes clinical illness within 2-4 days. At the doses tested, concurrent treatment of infected mice with tetracycline and the CPP-anti-tetA PNA or with the CPP-Sal-tsf PNA alone did not have a protective effect. Final conclusions are 1) that further research with the OMCs should focus on compounds with an Ir center and an amino acid ligand, and should explore ways to enhance intracellular penetration, 2) that the in vitro results of the PNA studies suggest that PNAs targeting expression of antibiotic resistance genes could allow for repurposing of antibiotics to which bacteria are resistant, and 3) additional study of the behavior of PNAs in vivo is advised. / Doctor of Philosophy / Antibiotic-resistant bacteria are increasingly recognized as a threat to global health, and new antibacterial drugs are urgently needed. Before a chemical compound can advance far in the journey to becoming a new drug it must be tested for toxicity against mammalian cells. A portion of this dissertation research involved testing the toxicity of several organometallic compounds (OMCs) previously shown to have antibacterial potential. Mouse-derived mammalian cells were treated with several of the OMCs, and initial results indicated that one of the OMCs is non-toxic and is likely a safe option for additional analysis. This OMC was further tested to see if it could inhibit mycobacterial growth inside of the mammalian cells. It did not effectively clear bacteria from inside of the mammalian cells, likely because of poor penetration of the cell membrane. Further research with this compound should focus on ways to effectively transport the OMC inside infected mammalian cells so that it can reach the bacteria it is meant to target. A second portion of this research involved using a peptide nucleic acid (PNA) to try and reverse tetracycline antibiotic resistance in the bacterial strain Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Peptide nucleic acids are short linear molecules that can bind strongly to complementary DNA and RNA sequences and thus be used to interfere with expression of specific genes. A PNA was designed to inhibit expression of the bacterial tetA gene that codes for a protein called the TetA tetracycline efflux pump, which imparts resistances to tetracycline. Treating the bacteria with the PNA resulted in a lower dose of tetracycline needed to inhibit bacterial growth, indicating a successful increase in tetracycline susceptibility. By using a molecular analysis technique called reversetranscriptase quantitative polymerase chain reaction (RT-qPCR), it was possible to measure the amount of tetA messenger RNA (mRNA) in cultures of DT104 treated only with tetracycline or with a combination of tetracycline and the PNA. As expected, bacteria treated with both the antibiotic and the PNA had less tetA mRNA than the cultures treated only with tetracycline, supporting the hypothesis that the PNA prevents the bacteria from effectively expressing the tetA gene. The PNA was next used in conjunction with tetracycline as an experimental treatment for mice infected with DT104. The PNA did not provide the expected protective effect under these circumstances. The overall conclusion for this part of the research is that PNAs offer an exciting potential avenue for counteracting antibiotic resistance, but additional experimentation is needed. Future research should focus on investigating more effective ways to get the PNAs inside the bacteria and on understanding more about how the PNAs behave in live animals. Several other PNAs targeting different genes involved in antibiotic resistance or essential bacterial functions were also tested against DT104 with variable success.
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Desarrollo de métodos moleculares de detección de virus de RNA de cultivos hortícolasRangel Aranguren, Ezequiel Alonzo 30 November 2015 (has links)
Uno de los problemas a los que se enfrenta la agricultura son los daños
producidos por los virus que causan cada año considerables pérdidas
económicas en diversos cultivos de todo el mundo. El control de las
enfermedades virales es difícil debido a su compleja y dinámica epidemiología,
con casos frecuentes de emergencia y rápida dispersión a escala global, así
como por su gran capacidad evolutiva que les permite superar distintas
estrategias de control, como por ejemplo el cultivo de variedades resistentes
obtenidas por mejora genética.
El primer paso para evaluar el estado sanitario de un cultivo y poder
aplicar un programa de manejo integrado es disponer de métodos de
diagnóstico y detección de los principales virus que sean específicos, fiables,
rápidos y sensibles. Dada la situación dinámica de las virosis, en la que
continuamente están apareciendo nuevos virus o variantes, es recomendable
que los métodos de diagnóstico puedan desarrollarse rápidamente para
responder a la demanda y que sean flexibles de manera que se pueda ajustar
al nivel de especificidad requerido (no solamente para la especie viral sino
también para categorías taxonómicas superiores, como género y familia o
inferiores como cepa o grupo de aislados virales). En este respecto, los
métodos moleculares basados en la reacción en cadena de la polimerasa
(polymerase chain reaction, PCR) y la hibridación molecular de ácidos
nucleicos cumplen estos criterios. Los métodos de PCR tienen la ventaja de ser
muy sensibles mientras que los basados en la hibridación permiten analizar
simultáneamente un elevado número de muestras. Una modalidad, la
hibridación de flujo de improntas vegetales (flow-through hybridization of
tissue prints), recientemente desarrollada, supone una reducción drástica del
tiempo necesario para el análisis al evitar el procesamiento de las muestras y
acelerar el proceso de hibridación. En el desarrollo, tanto de la PCR como de la
hibridación, es necesario conocer la secuencia nucleotídica de al menos una
porción del genoma del virus que se quiere detectar, pero si se quiere sacar el
máximo provecho, por una parte, hay que estimar la variabilidad genética
entre los distintos aislados del virus, para así evitar o minimizar los falsos
negativos (ej. reacción negativa con algunos aislados genéticamente
divergentes), y por otra parte, hay que considerar la relación genética del virus
objeto con repecto a otros virus, para evitar falsos positivos (ej. reacción
positiva con aislados pertenecientes a otros virus).
En esta tesis doctoral se abordaron dos retos relacionados con el
desarrollo de métodos moleculares de detección de ribovirus (con genoma de
RNA) que afectan a cultivos hortícolas. El primero corresponde a la detección
del género Fabavirus, que está compuesto por cinco especies virales: virus 1
del marchitamiento del haba (Broad bean wilt virus 1, BBWV-1), virus 2 del
marchitamiento del haba (Broad bean wilt virus 2, BBWV-2), virus del mosaico
de la genciana (Gentian mosaic virus, GeMV) y virus del mosaico suave de
cucurbitáceas (Curcubit mild mosaic virus, CuMMV) y virus del mosaico suave
de la ortiga blanca (Lamium mild mosaic virus, LMMV). En primer lugar se
secuenció el genoma completo del único aislado disponible de LMMV, ya que
era la única especie viral de este género de la que no se disponía ninguna
secuencia nucleotídica. La comparación de esta secuencia con la del genoma
completo de los otros fabavirus confirmó que LMMV es una especie viral per
se al ajustarse a los criterios actualmente aceptados de demarcación entre
generos, especies y aislados virales. Se encontró varias repeticiones de un
motivo de diez nucleotídos en las zonas no traducibles del extremo 5’ de todos
los miembros del género Fabavirus que se puede considerar como una seña de
identidad y serviría para asignar nuevos virus a este género viral. En segundo
lugar, a partir de todas las secuencias nucleotídicas disponibles de todos los
virus del género Fabavirus se diseñó una pareja de iniciadores conservados
para poder detectar por retrotranscripción (reverse transcription, RT) y PCR
(RT-PCR) todos los fabavirus en una única reacción. También se diseñaron
cinco parejas de iniciadores, cada una específica para una especie viral, para
poder detectar e identificar cualquiera de estos virus en una única reacción
mediante RT-PCR múltiple (multiplex RT-PCR). La combinación de ambos
procedimientos de RT-PCR permite detectar e identificar cada una de las cinco
especies conocidas del género Fabavirus, así como descubrir nuevas especies
dentro de este género. En tercer lugar, se diseñó una sonda molecular
correspondiente a la región del genoma que contenia las repeticiones del
motivo de diez nucleótidos, con la que se pudo detectar las cinco especies del
género Fabavirus mediante hibridación molecular de flujo. Esta es la la
primera vez que se consigue una sonda conservada para un género viral.
También se diseñaron cinco sondas, cada una específica para cada una de
cinco las especies conocidas del género. De manera que mediante hibridación
de flujo con las seis sondas se pueden identificar BBWV-1, BBWV-2, LMMV,
GeMV y CuMMV y descubrir nuevas especies dentro del género Fabavirus.
Para comprobar la especificidad de las técnicas de RT-PCR e hibridación
desarrolladas aquí, ambas se probaron con distintos aislados de una misma
especie viral que eran genéticamente muy divergentes (identidad ~80%) y no se
produjeron falsos negativos. Así mismo se constató in silico o in vitro que estas
técnicas no producían falsos positivos al no detectar otros virus relacionados
fuera del género Fabavirus, como son aquellos que pertenecen a los géneros
Comovirus y Nepovirus, que junto el género Fabavirus forman la subfamilia
Comovirinae.
El otro reto que se planteó fue la detección e identificación de los siete
ribovirus más importantes en los cultivos de tomate del área mediterránea y
otras áreas subtropicales: virus del mosaico del pepino (Cucumber mosaic
virus, CMV), virus del bronceado del tomate (Tomato spotted wilt virus, TSWV),
virus del mosaico del tomate (Tomato mosaic virus, ToMV), virus del la clorosis
del tomate (Tomato chlorosis virus,ToCV), virus de la clorosis infecciosa del
tomate (Tomato infectious clorosis virus, TICV), virus del mosaico del pepino
dulce (Pepino mosaic virus, PepMV), y virus del torrado del tomate (Tomato
torrado virus, ToTV). Primero se caracterizó la variabilidad genética de ToMV
puesto que era el único de estos virus en la que no se había estudiado este
atributo. Se encontró tres grupos de aislados que tenían entre ellos
identidades nucleotídicas menores al 90%. Sin embargo, solamente uno de
estos grupos se había dispersado por el mundo y se caracterizaba por una
gran estabilidad y muy baja variabilidad genética (identidades mayores del
98%). A partir del análisis de todas las secuencias disponibles de estos siete
virus se diseñaron siete parejas de iniciadores específicos para cada virus. Se
tuvo en cuenta la variabilidad genética dentro de cada uno de los siete virus
para evitar o minimizar los falsos negativos. Mediante dos RT-PCR múltiples
(una para CMV, ToMV y TICV y la otra para TSWV, ToCV, ToTV y PepMV) se
pudo detectar e identificar cada especie viral tanto en infecciones únicas como
múltiples en muestras de campo de Sicilia con una prevalencia variable según
el virus y el área geográfica.
Los métodos moleculares de detección desarrollados en esta tesis
permiten llevar a cabo prospecciones rutinarias en grandes áreas de cultivo,
recabando información epidemiológica muy valiosa para el manejo de
enfermedades a diferentes escalas geográficas, y constituyen una primera
línea de defensa frente a la ocurrencia de brotes de las enfermedades virales.
El desarrollo y adopción de esta metodología supone un aporte que
contribuiría a mejorar la gestión de la calidad por parte de empresas
productoras y distribuidoras de semillas o propágulos asexuales, y a las
agencias gubernamentales reguladoras del comercio internacional de estos
productos con la finalidad de disminuir los riesgos y reducir el enorme
impacto económico, valorado en decenas de millones de euros anuales, en
pérdidas directas e indirectas, que implica la emergencia y dispersión de los
virus en las economías de los países afectados. / Rangel Aranguren, EA. (2015). Desarrollo de métodos moleculares de detección de virus de RNA de cultivos hortícolas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58269
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Développement d'une méthodologie PCR en temps réel pour la détection et la quantification in planta des principaux champignons pathogènes associés aux maladies du bois de la vigne / Development of a real time PCR methodology for in planta detection and quantification of the main fungal pathogens associated with grapevine trunk diseasesPouzoulet, Jérôme 13 July 2012 (has links)
Les maladies fongiques du bois de la vigne que sont le syndrome de l'esca, le Black Dead Arm (BDA) et l'Eutypiose sont particulièrement dommageables à la profession vitivinicole, et sont actuellement en progression. Le temps d'incubation nécessaire à l'expression de ces maladies au champ complique l'évaluation de solutions préventives adaptées en condition contrôlée ainsi qu'en condition de terrain. Ces travaux de thèse ont eu pour objectifs la conception et la validation de tests PCR quantitatifs en temps réel (RtqPCR), permettant la détection et la quantification in planta de cinq champignons associés aux maladies de dépérissement de la vigne, Phaeomoniella chlamydospora et Phaeoacremonium aleophilum (esca), Diplodia seriata et Neofusicoccum parvum (BDA), et Eutypa lata (Eutypiose). Le développement de tests multiplexes a ensuite été entrepris et ces derniers ont été évalués pour la détection de quatre champignons (2 associés à esca et 2 au BDA) dans le bois de jeunes plants issus de pépinière viticole. Enfin, l'étude de l'interaction in planta de deux champignons associés au syndrome de l'esca de la vigne (P.chlamydospora et P.aleophilum) a été réalisée par RT-qPCR, et complétée par la caractérisation histologique de la réponse de la plante à la blessure dans le bois, en inoculation individuelle et en co-inoculation. / Grapevine trunk diseases, among which esca's syndrome, Black Dead Arm (BDA) and Eutypiosis, represent a real threat for grape and wine industry. Incubation time required before symptoms externalization in field complicates the evaluation of the efficacy of preventive solution in control and field conditions. These thesis's works focused on the design and the validation of Real Time quantitative PCR assays (RT-qPCR), in order to detect and quantify in vine plant five fungi associated with grapevine trunk disease, Phaeomoniella chlamydospora et Phaeoacremonium aleophilum (esca), Diplodia seriata et Neofusicoccum parvum (BDA), et Eutypa lata (Eutypiosis). The development of multiplex assays was undertaken and these last were evaluated in order to detect four fungi (esca and BDA) in wood sample from young plants in vine nursery. Finally, a study of the interaction between two fungi associated with esca's syndrome has been determined in planta through RT-qPCR, and completed by a histological analysis of plant response to injury of woody tissues.
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Localização dos transcritos dos genes WNT5A e HOXB5 em carcinomas epidermóides de boca através da técnica de hibridização in situ. / WNT5A e HOXB5 localization study in oral squamous cell carcinoma with in situ hybridization.Almeida, Fernanda Campos Sousa de 10 December 2004 (has links)
São freqüentes alterações em vias de sinalização de genes de desenvolvimento, no que diz respeito ao carcinoma epidermóide de boca (CEB). Vinte e nove casos de CEB e tecido não tumoral adjacente à neoplasia foram investigados através da técnica de hibridização in situ". Os transcritos dos genes WNT5A e HOXB5 foram observados em todos os casos de tumor. A hibridização in situ" revelou que o WNT5A estava mais expresso em tumores bem diferenciados. Adicionalmente, observou-se transcritos do WNT5A em glândulas salivares menores, estroma glandular, estroma tumoral e alguns vasos sanguíneos Entretanto, com respeito ao HOXB5 não foi possível estabelecer mudança do padrão do transcrito nos diferentes graus histológicos nas amostras de CEB. O HOXB5 também pôde ser identificado em alguns fragmentos de glândulas salivares menores, tecido muscular e em endotélio. Os resultados do presente estudo sugerem que a expressão dos genes WNT5A e HOXB5 podem estar relacionadas com a diferenciação e progressão do câncer de boca. / Disruption in developmental genes pathway are common in oral squamous cell carcinoma (OSCC). This study investigated the pattern of expression of two developmental genes, WNT5A and HOXB5, in 29 cases of OSCC and adjacent non tumoural tissue using in situ hybridization technique. Transcripts for WNT5A and HOXB5 were detected in all tumoral samples. In situ hybridization technique demonstrated that WNT5A transcripts were mainly detected in well differentiated tumors when compared with moderately and undifferentiated OSCC. WNT5A transcripts were also observed in accessory salivary glands, glandular stroma, and vessels. Therefore, for the HOXB5 transcript it was not possible to stability a relationship with the tumoral histological grade. The expression of HOXB5 transcripts in non tumoral samples was detected in salivary glands, glandular stroma, endothelium, and muscle. Results suggest that WNT5A and HOXB5 genes play a possible role in tumor differentiation and cancer progression.
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Detecção e tipificação do vírus da dengue por RT-PCR em tempo real / Detection and typing of dengue virus by real time RT-PCR assaysPoloni, Telma Regina Ramos Silva 28 May 2009 (has links)
A dengue é uma doença infecciosa de transmitida pela picada de mosquitos do gênero Aedes. O vírus da dengue (DENV), pertencente ao gênero Flavivirus, família Flaviviridae, é atualmente um importante problema de saúde pública em todo o mundo. São reconhecidos quatro sorotipos antigenicamente distintos (DENV-1, -2, -3 e -4). A doença causada por qualquer um dos sorotipos cursa de forma assintomática ou com quadro clínico que varia desde uma febre indiferenciada e autolimitada, passando pela febre clássica da dengue (FD) até quadros graves de febre hemorrágica da dengue (DHF). O diagnóstico clínico é difícil de ser realizado principalmente na fase aguda da doença em que os sintomas são muito similares aos de outras infecções febris agudas, ficando a cargo do laboratório o diagnóstico definitivo. Os métodos sorológicos para detecção de anticorpos IgM/IgG são os mais amplamente utilizados, mas inadequados para diagnóstico precoce uma vez que detectam anticorpos a partir do sexto dia do início dos sintomas. Os métodos moleculares estão sendo cada vez mais utilizados para o diagnóstico precoce por serem mais rápidos e sensíveis que a sorologia e o isolamento viral. Neste estudo comparamos a sensibilidade de uma RT-PCR em tempo real gênero específica com um ELISA para detecção da proteína NS1 comercialmente disponível analisando amostras de soro de pacientes com dengue. Também foram desenvolvidos dois protocolos de RT-PCR em tempo real para identificação do sorotipo viral, uma contendo primers para extremidade 5 do genoma viral e outra contendo primers para a região codificadora da proteína NS5. A RT-PCR em tempo real gênero específica mostrou-se mais sensível que o ELISA, principalmente nas amostras que apresentavam baixa carga viral. A RT-PCR em tempo real contendo os primers para a extremidade 5 apresentou uma sensibilidade baixa quando comparada com a RT-PCR genérica, porém foi mais sensível que aquela contendo os primers para a região codificadora da proteína NS5. Considerando os resultados obtidos, sugerimos uma estratégia de triagem dos casos suspeitos de dengue utilizando a RT-PCR genérica para posteriormente identificar o sorotipo viral com o protocolo que utiliza os primers da extremidade 5. Embora este último protocolo tenha sido pouco sensível, a identificação do sorotipo infectante em algumas amostras é suficiente para definir qual o sorotipo circulante durante uma epidemia. / Dengue is an infectious disease transmitted by the biting of mosquitoes of Aedes genus. Dengue virus (DENV), belonging to the Flavivirus genus, Flaviviridae family, is an important public health problem worldwide. Four antigenically distinct viruses are recognized (DENV-1, -2, -3, e -4). Infection with any of the virus serotypes causes a spectrum of the illness ranging from inapparent or mid viral syndrome to classic dengue fever (DF) and severe hemorrhagic disease (DHF). The clinical diagnosis is difficult especially in the acute phase of the disease when the symptoms are very similar to other febrile illness, corresponding to the laboratory the definitive diagnosis. Serological methods detecting antibodies IgM/IgG are the more widely used; however, they are inappropriate for early diagnosis since these methods detect antibodies after six day of the onset of symptoms. The molecular methods are more frequently used for the early diagnosis because they are faster and more sensitive than serological methods and virus isolation. In this study, we have compared the sensitivity of a generic real time RT-PCR with a commercial ELISA for the NS1 protein analyzing serum samples from patients with dengue virus infection. We have also developed two protocols of real time RT-PCR to identify dengue serotype, one of them containing primers to the 5 end and the other, primers to the NS5 coding region. The generic real time RT-PCR showed to be more sensitive than the ELISA, principally, between the samples with low viral load. The real time RT-PCR containing primers to the 5 end showed a lower sensitivity than the generic real time RT-PCR; however, it was more sensitive than that containing primers to the NS5 coding region. Considering these results, we suggest the use of the generic real time RT-PCR to screen the dengue suspected cases and then to identify the serotype using the protocol that include the primers to the 5 end. Although the last protocol has shown a low sensitive, the identification of the infecting serotype in some of the samples is enough to define which serotype is circulating during the epidemic period.
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