Comparison of four methods of genotyping IL28B polymorphisms in chronic hepatitis C patients.

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 (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.

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.