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

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

Author Arraes, Luiz Claudio Autor UNIFESP Google Scholar
Souza, Paulo Roberto de Google Scholar
Bruneska, Danielly Google Scholar
Castelo Filho, Adauto Autor UNIFESP Google Scholar
Cavada, Benildo de Sousa Google Scholar
Lima Filho, José Luiz de Google Scholar
Crovella, Sergio Google Scholar
Institution Universidade Federal de Pernambuco Laboratório de Imunopatologia Keizo Asami
Instituto Materno Infantil de Pernambuco
Universidade Federal de São Paulo (UNIFESP)
Universidade Federal do Ceará Departamento de Bioquímica e Biologia Molecular
University of Trieste Department of Developmental and Reproductive Sciences
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.
Keywords Melting temperature assay
Real time PCR
MBL2
Polymorphisms
HIV-1-positive children
Language English
Date 2006-06-01
Published in Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 39, n. 6, p. 719-723, 2006.
ISSN 0100-879X (Sherpa/Romeo, impact factor)
Publisher Associação Brasileira de Divulgação Científica
Extent 719-723
Origin http://dx.doi.org/10.1590/S0100-879X2006000600003
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000238305200003
SciELO ID S0100-879X2006000600003 (statistics in SciELO)
URI http://repositorio.unifesp.br/handle/11600/3061

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