title: A cost-effective melting temperature assay for the detection of single-nucleotide polymorphism in the MBL2 gene of HIV-1-infected children.
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 60 degrees to 95 degrees C in 0.2 degrees C 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.
authors.affiliation: Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Recife, PE, Brasil.
authors.forename: L C
authors.initials: LC
authors.lastname: Arraes
authors.forename: P R
authors.initials: PR
authors.lastname: de Souza
authors.forename: D
authors.initials: D
authors.lastname: Bruneska
authors.forename: A
authors.initials: A
authors.lastname: Castelo Filho
authors.forename: B de Souza
authors.initials: Bde S
authors.lastname: Cavada
authors.forename: J L
authors.initials: JL
authors.lastname: de Lima Filho
authors.forename: S
authors.initials: S
authors.lastname: Crovella
mesh_terms: D016022:Case-Control Studies; D002648:Child; D003362:Cost-Benefit Analysis; D005787:Gene Frequency; D015658:HIV Infections / Q000235:genetics* / Q000635:transmission; D006801:Humans; D037601:Mannose-Binding Lectin / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D013696:Temperature
chemical_list: C445619:MBL2 protein, human; D037601:Mannose-Binding Lectin