A cost-effective melting temperature assay for the detection of single-nucleotide polymorphism in the MBL2 gene of HIV-1-infected children

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dc.contributor.author Arraes, Luiz Claudio [UNIFESP]
dc.contributor.author Souza, Paulo Roberto de
dc.contributor.author Bruneska, Danielly
dc.contributor.author Castelo Filho, Adauto [UNIFESP]
dc.contributor.author Cavada, Benildo de Sousa
dc.contributor.author Lima Filho, José Luiz de
dc.contributor.author Crovella, Sergio
dc.date.accessioned 2015-06-14T13:32:06Z
dc.date.available 2015-06-14T13:32:06Z
dc.date.issued 2006-06-01
dc.identifier http://dx.doi.org/10.1590/S0100-879X2006000600003
dc.identifier.citation Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 39, n. 6, p. 719-723, 2006.
dc.identifier.issn 0100-879X
dc.identifier.uri http://repositorio.unifesp.br/handle/11600/3061
dc.description.abstract We report a fast (less than 3 h) and cost-effective melting temperature assay method for the detection of single-nucleotide polymorphisms in the MBL2 gene. The protocol, which is based on the Corbett Rotor Gene real time PCR platform and SYBR Green I chemistry, yielded, in the cohorts studied, sensitive (100%) and specific (100%) PCR amplification without the use of costly fluorophore-labeled probes or post-PCR manipulation. At the end of the PCR, the dissociation protocol included a slow heating from 60º to 95ºC in 0.2ºC steps, with an 8-s interval between steps. Melting curve profiles were obtained using the dissociation software of the Rotor Gene-3000 apparatus. Samples were analyzed in duplicate and in different PCR runs to test the reproducibility of this technique. No supplementary data handling is required to determine the MBL2 genotype. MBL2 genotyping performed on a cohort of 164 HIV-1-positive Brazilian children and 150 healthy controls, matched for age and sex and ethnic origin, yielded reproducible results confirmed by direct sequencing of the amplicon performed in blind. The three MBL2 variants (Arg52Cys, Gly54Asp, Gly57Glu) were grouped together and called allele 0, while the combination of three wild-type alleles was called allele A. The frequency of the A/A homozygotes was significantly higher among healthy controls (0.68) than in HIV-infected children (0.55; P = 0.0234) and the frequency of MBL2 0/0 homozygotes was higher among HIV-1-infected children than healthy controls (P = 0.0296). The 0 allele was significantly more frequent among the 164 HIV-1-infected children (0.29) than among the 150 healthy controls (0.18; P = 0.0032). Our data confirm the association between the presence of the mutated MBL2 allele (allele 0) and HIV-1 infection in perinatally exposed children. Our results are in agreement with the literature data which indicate that the presence of the allele 0 confers a relative risk of 1.37 for HIV-1 infection through vertical transmission. en
dc.format.extent 719-723
dc.language.iso eng
dc.publisher Associação Brasileira de Divulgação Científica
dc.relation.ispartof Brazilian Journal of Medical and Biological Research
dc.rights Acesso aberto
dc.subject Melting temperature assay en
dc.subject Real time PCR en
dc.subject MBL2 en
dc.subject Polymorphisms en
dc.subject HIV-1-positive children en
dc.title A cost-effective melting temperature assay for the detection of single-nucleotide polymorphism in the MBL2 gene of HIV-1-infected children en
dc.type Artigo
dc.contributor.institution Universidade Federal de Pernambuco Laboratório de Imunopatologia Keizo Asami
dc.contributor.institution Instituto Materno Infantil de Pernambuco
dc.contributor.institution Universidade Federal de São Paulo (UNIFESP)
dc.contributor.institution Universidade Federal do Ceará Departamento de Bioquímica e Biologia Molecular
dc.contributor.institution University of Trieste Department of Developmental and Reproductive Sciences
dc.description.affiliation Universidade Federal de Pernambuco Laboratório de Imunopatologia Keizo Asami
dc.description.affiliation Instituto Materno Infantil de Pernambuco
dc.description.affiliation Universidade Federal de São Paulo (UNIFESP) Disciplina de Gastroenterologia
dc.description.affiliation Universidade Federal do Ceará Departamento de Bioquímica e Biologia Molecular
dc.description.affiliation University of Trieste Department of Developmental and Reproductive Sciences
dc.description.affiliationUnifesp UNIFESP, Disciplina de Gastroenterologia
dc.identifier.file S0100-879X2006000600003.pdf
dc.identifier.scielo S0100-879X2006000600003
dc.identifier.doi 10.1590/S0100-879X2006000600003
dc.description.source SciELO
dc.identifier.wos WOS:000238305200003



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