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https://www.arca.fiocruz.br/handle/icict/12054
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2016-07-31
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- IOC - Artigos de Periódicos [12125]
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COMPARISON OF FOUR METHODS OF GENOTYPING IL28B POLYMORPHISMS IN CHRONIC HEPATITIS C PATIENTS
Affilliation
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Abstract
Background: Single nucleotide polymorphisms (SNPs) of the interleukin 28B (IL28B) gene are associated
with viral clearance and treatment response in hepatitis C virus (HCV) infection; however, most of the
available SNP genotyping methods are expensive.
Aims: This study sought to evaluate the cost effectiveness of four methods used to genotype the
rs12979860 and rs8099917 SNPs of the IL28B gene.
Methods: Tetra-primer amplification-refractory mutation system-polymerase chain reaction (ARMSPCR),
restriction fragment length polymorphism (RFLP), quantitative (q) PCR and direct sequencing
methods were evaluated in terms of specificity, cost and run time in 281 blood samples obtained from
chronic HCV patients.
Results: In ARMS-PCR method, the primers designed to target both SNPs produced PCR fragments of
specific sizes that distinguished the alleles of rs12979860 and rs8099917. In RFLP, the band profile
allowed the distinction between genotypes. The qPCR was the faster and easier to perform. Validation by
nucleotide sequencing showed 100% agreement among the three methods. The cost for a single reaction
was lowest for ARMS-PCR, followed in turn by RFLP, qPCR and sequencing.
Conclusions: The methodology described for the ARMS-PCR showed the most favorable cost–benefit ratio.
Moreover, this approach is fast and simple, requiring only equipment that is commonly used in molecular
diagnosis, which is an essential parameter for use in developing countries where laboratories have scarce
financial resources.
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