P035 A simple and reliable method for genotyping IL28B polymorphism in patients infected with hepatitis C virus

Abstract

Introduction The standard of cure for hepatitis C virus (HCV) infection is a combination of Peg-IFNα with Ribavirin. However, about 50% of the patients infected with HCV genotype 1 achieved sustained virologic response (SVR). Several single nucleotide polymorphisms (SNP) near IL28B gene (encoding IFN-λ3), including SNP rs12979860, were shown to be linked with SVR [1] , [2] , [3] . For the genotyping of the IL28B polymorphism, several methods have been developed [4] , [5] , [6] , [7] . However, these methods require specific instrument and are quite expensive. Therefore, it is suitable to establish a method for routine practice. Here, we present a simple and reliable method based on polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis for genotyping rs12979860 of IL28B. Methods Serum samples and liver biopsies were collected from 35 HCV infected patients. DNA was extracted from serum samples using High Pure PCR Template Preparation Kit (Roche) and from liver specimens using QIAamp DNA FFPE Tissue Kit (Qiagen). Primers were designed from the sequence surrounding rs12979860 to be used in nested PCR: first round primers were 5′-CACTCCCGGGCCTCACCGAT-3′ (Sense), and 5′-ACCACGAGACCCCCGCAGTC-3′ (Antisense); second round primers were: 5′-GCTTATCGCATACGGCTAGG-3′ (Sense), and 5′-AGGCTCAGGGTCAATCACAG-3′ (Antisense). PCR were performed on 2700 PCR machine (Applied BioSystems) by 40 cycles (94 °C 30 s, 58 °C 30 s, 72 °C 30 s). For RFLP analysis, BstUI enzyme was selected on basis of specific cut of alleles of rs 12979860. Aliquots of the second PCR products were digested with BstUI enzyme (Biolabs), fragments were separated on a 2% agarose gel electrophoresis. To validate method, all genotyping data were compared with those obtained with LightMix® Kit IL28B (TIB MOLBIOL) and PCR products were sequenced. Statistical analyses were performed by Mann-Withney U test (p Results The final PCR product containing the SNP rs12979860 is 242 bp long. The specificity of the PCR was confirmed by sequencing of the products. For RFLP, BstUI enzyme cuts PCR products generating fragments of 135, 82, and 25 bp for C allele or of 160 and 82 bp for T allele. All identifications require BstUI cuts that can be easily checked. The allelic distribution in our survey was 14/35 (40%) for CC, 10/35 (28%) for CT and 11/35 (31%) for TT genotypes. All typing data achieved by our PCR-RFLP method were identical to those obtained with LightMix kit and concordant with direct DNA sequencing results. Conclusion We have developed successfully genotyping assay based on PCR-RFLP for the IL28B polymorphism (rs12979860). The greatest advantages of this method reside in its simplicity, reliability and low cost, thus providing a suitable alternative for routine diagnostics in a conventional laboratory without required substantial equipment investment.