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 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. Correspondence

Mannose binding lectin gene polymorphisms are associated with type 1 diabetes in Brazilian children and adolescents

“…MBL2 allelic and genotype frequencies obtained in our groups of patients and controls were comparable to those reported in two previous studies performed on HIV vertical transmission in children populations also coming from Recife [20,21].…”

“…We screened for MBL2 functional polymorphisms 214 children and adolescents with prior diagnosis of type 1 diabetes and 214 healthy controls, all coming from Recife, by using a rapid and low-cost genotyping technique based on the melting temperature assay [19,20].…”

Association of hepatitis C virus infection and liver fibrosis severity with the variants alleles of MBL2 gene in a Brazilian population

“…Melting curve profiles were obtained using the dissociation software of the Rotor Gene-6000. All three variant alleles of exon 1 were grouped as allele O, whereas the wild-type allele was called A [19,20].…”

High-throughput genotyping of mannose-binding lectin variants using high-resolution DNA-melting analysis

“…Assays have been developed for MBL2 genotyping in the past employing amplicon melting curve analysis with SybrGreenI (Hladnik et al, 2002;Arraes et al, 2006) and fluorescent hybridization probes (Steffensen et al, 2003). While fluorescent hybridization probes (Steffensen et al, 2003) in combination with HRMA should be able to resolve all MBL2 variants, this method uses expensive fluorophores.…”