Delvaux Nathália, da Costa Vanessa Duarte, da Costa Maristella Matos, Lampe Elisabeth
Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Avenida Brasil, 4365 - Manguinhos, Código, 21040-900 Rio de Janeiro, RJ, Brazil.
Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Avenida Brasil, 4365 - Manguinhos, Código, 21040-900 Rio de Janeiro, RJ, Brazil.
J Virol Methods. 2015 Aug;220:1-4. doi: 10.1016/j.jviromet.2015.04.001. Epub 2015 Apr 9.
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.
This study sought to evaluate the cost effectiveness of four methods used to genotype the rs12979860 and rs8099917 SNPs of the IL28B gene.
Tetra-primer amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR), 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.
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.
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.
白细胞介素28B（IL28B）基因的单核苷酸多态性（SNP）与丙型肝炎病毒（HCV）感染中的病毒清除和治疗反应相关；然而，大多数现有的SNP基因分型方法成本高昂。
本研究旨在评估用于对IL28B基因的rs12979860和rs8099917 SNPs进行基因分型的四种方法的成本效益。
在从慢性HCV患者获得的281份血液样本中，从特异性、成本和运行时间方面对四引物扩增阻滞突变系统-聚合酶链反应（ARMS-PCR）、限制性片段长度多态性（RFLP）、定量（q）PCR和直接测序方法进行了评估。
在ARMS-PCR方法中，针对两个SNP设计的引物产生了特定大小的PCR片段，可区分rs12979860和rs8099917的等位基因。在RFLP中，条带图谱可区分基因型。qPCR执行起来更快且更简便。通过核苷酸测序进行的验证显示三种方法之间的一致性为100%。ARMS-PCR单次反应的成本最低，其次依次是RFLP、qPCR和测序。
所描述的ARMS-PCR方法显示出最有利的成本效益比。此外，这种方法快速且简单，仅需要分子诊断中常用的设备，这对于实验室资金匮乏的发展中国家来说是一个关键参数。
