942 resultados para nested-PCR_


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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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Pós-graduação em Medicina Veterinária - FMVZ

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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The Goliath grouper (Epinephelus itajara) is one of the most endangered species of fish of the subfamily Epinephelinae. Slow to develop and mature, and dependent on mangrove habitats for breeding, the species also suffers intense harvesting, which has reduced drastically in numbers in many areas. To contribute to the understanding of the characteristics of E. itajara populations, we conducted a molecular genetics study of the species, focusing on populations from the Northern Brazilian coast. The mtDNA control region (D-loop) of 116 individuals from five localities (Bragança, Ajuruteua, Parnaíba, Fortaleza and Natal) was analysed, and a sequence of 499 base pairs identified. Analyses of the sequences indicated that genetic variability was generally lower in E. itajara than in other endangered species of the genus. AMOVA found no significant grouping structure among the populations. Nested Clade Analysis revealed a significant association between genetic variability and geographic distribution among only three populations (Ajuruteua, Parnaíba and Natal). Genetic diversity was higher in populations from the Amazon region, which may be related to the better conservation of mangrove habitats in this area. Therefore, the present study could be used for the implementation of conservation and management measures in order to protect and consolidate these populations.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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ABSTRACT: Antibodies to human T-cell lymphotropic virus-1 and 2 (HTLV-1 and 2) were tested in 259 inhabitants (98 males and 161 females) of four villages of the Marajó Island (Pará, Brazil) using enzyme immunoassays (ELISA and Western blot). Types and subtypes of HTLV were determined by nested polymerase chain reaction (PCR) targeting the pX, env and 5´LTR regions. HTLV-1 infection was detected in Santana do Arari (2.06%) and Ponta de Pedras (1%). HTLV-2 was detected only in Santana do Arari (1.06%). Sequencing of the 5´LTR region of HTLV-1 and the phylogenetic analysis identified the virus as a member of the Cosmopolitan Group, subgroup Transcontinental. Santana do Arari is an Afro-Brazilian community and the current results represent the first report of HTLV-1 infection in a mocambo located in the Brazilian Amazon region.

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ABSTRACT: The present work evaluated the epidemiology of human immunodeficiency virus 1/human T-cell lymphotropic virus (HIV-1/HTLV) coinfection in patients living in Belém (state of Pará) and Macapá (state of Amapá), two cities located in the Amazon region of Brazil. A total of 169 blood samples were collected. The sera were tested by enzyme-linked immunosorbent assay to determine the presence of antibodies anti-HTLV-1/2. Confirmation of infection and discrimination of HTLV types and subtypes was performed using a nested polymerase chain reaction targeting the pX and 5' LTR regions, followed by restriction fragment length polymorphism and sequencing analysis. The presence of anti-HTLV1/2 was detected in six patients from Belém. The amplification of the pX region followed by RFLP analysis, demonstrated the presence of HTLV-1 and HTLV-2 infections among two and four patients, respectively. Sequencing HTLV-1 5' LTR indicated that the virus is a member of the Cosmopolitan Group, Transcontinental subgroup. HTLV-2 strains isolated revealed a molecular profile of subtype HTLV-2c. These results are a reflex of the epidemiological features of HIV-1/HTLV-1/2 coinfection in the North region of Brazil, which is distinct from other Brazilian regions, as reported by previous studies.

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ABSTRACT: Human T-lymphotropic virus tipe 1 is recognized as the etiologic agent of tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). A very similar clinical disease has been increasingly associated to HTLV-2, whose pathogenicity still requires further assessments. This transversal, retrospective epidemiological survey aimed to determine the prevalence of HTLV among individuals with neurological disturbances and further evaluate cases of inconclusive serology using molecular biology methods. The present study involved patients inhabitants of Pará State and/or admitted at health institutions of the and who were referred to the Virology Section of Instituto Evandro Chagas (IEC) by local doctors between January of 1996 and December 2005, to search for the presence of HTLV-1/2 serum antibodies. Of these patients 353 were selected, with age between 9 months and 79 years, who presented at least one signal or symptom of the Marsh’s Complex (1996), as well as had HTLV-1/2 positive serology at screening and confirmatory ELISA. The overall prevalence of HTLV antibodies by ELISA as 8,8% (31/353), with rates of 10,6% (19/179) and 6,9% (12/174) for female and male patients, respectively. Among HTLV-1/2 the 31 ELISA-positive patients it was noted that 15 (48.4%) of 31 had paresis (n = 8), parestesis (n = 5), and paraplegia (n = 3). Of these 31 HTLV ELISA positive patients, 25 could be submitted to WB for assessment of viral types, which were distributed as follow: 80% (20/25) were HTLV-1, 12% (3/25) were HTLV-2, one case was of HTLV-1+HTLV-2 infection (4%), and serum from one patient yielded an indeterminate profile (4%). Only 14 of these 25 patients could be re-localised for collection of an additional sample for molecular analysis. It was observed that 78.6% of samples typed by WB had the proviral TAX region successfully amplified by nested-PCR. In addition, types were confirmed as based on results obtained from the amplification of the POL region using real-time PCR; this denoted good specificity and sensitivity of the WB used in this study. The sample defined as HTLV-1+HTLV-2 infection by WB was amplified in its TAX region but real time PCR confirmed HTLV-1 infection only. The patient with WB indeterminate profile and one of samples typed as HTLV-2 by WB were amplified by nested-PCR but the real time PCR was negative for HTLV-1 and HTLV-2 in both samples. One patient presenting clinical manifestations of crural myalgia and parestesia with duration of about 7 years reacted HTLV-2-positive by both WB and real-time PCR, a denoting a clear HTLV-2- related chronic myelopathy. This study has identified a case of possible vertical transmission in two distinct situations: a patient whose mother presented antibodies for HTLV-1 by WB and two sisters who reacted HTLV-1-positive by WB and real-time PCR. Although of epidemiological relevance, results from this study warrant further and broader analyses concerning the molecular epidemiology of HTLV types and subtypes HTLV. In addition, a more complete clinical assessment of neurological symptoms should be further performed, in order to better characterise cases of HTLV-related chronic myelopathy in our region.