Computational protocol: The Prevalence of Hepatitis C Virus Core Amino Acid 70 Substitution and Genotypes of Polymorphisms Near the IFNL3 Gene in Iranian Patients With Chronic Hepatitis C

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Protocol publication

[…] Assessment of the HCV RNA level was carried out using COBAS® TaqMan® HCV Test v2.0 (Roche Diagnostics) according to the manufacturer’s instructions. Nested RT-PCR followed by DNA sequencing were used for the assessment of HCV core aa 70 substitutions. Briefly, viral RNA was extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The extracted viral RNA was subjected to cDNA synthesis using a RevertAid First Strand cDNA Synthesis Kit (Fermentas of Thermo Fisher Scientific, Waltham, MA, US). The nested RT-PCR was performed using an Accupower PCR PreMix (Bioneer Corp., Daejeon, South Korea) with the following conditions for both the first and nested rounds of PCR: 94°C for 5 minutes, 35 cycles at 94°C for 30 seconds, 50°C for 40 seconds, and 72°C for 40 seconds, followed by 72°C for 5 minutes. The primers were adopted from the study by Kumar et al. (). Primers used for the first round reaction included the following: HG1, 5’-ATGTACCCCATGAKRTCGGC-3’ and HG2, 5’-ACTGCCTGATAGGGTGCTTGCGAG-3’; the nested round reaction used the following primers: HG3, 5’-CAYGTRAGGGTATCGATGAC-3’ and HG4, 5’-AGGTCTCGTAGACCGTGCATCATG-3’. The 404 base pair (bp) amplified products of the nested RT-PCR were purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), followed by DNA sequencing with a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, US) and a 3130XL ABI Genetic Analyzer (Applied Biosystems, Foster City, CA, US). The sequence traces were assessed using a CLC Sequence Viewer (version 6), and the DNA sequences were translated to protein to detect any changes at the 70 position of the HCV core protein. The HCV genotyping was performed based on phylogenetic analysis using MEGA 5 software and the neighbor-joining tree and Kimura 2-parameter models (The phylogenetic tree will not be represented in the results section of this study). The rs12979860 and rs8099917 polymorphisms were assessed as the most common polymorphisms near the IFNL3 gene. The detailed protocol of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for genotyping the rs12979860 and rs8099917 polymorphisms was as previously described (). The latest laboratory data of the serum alanine aminotransferase (ALT) level (IU/L) was recruited from patient records. […]

Pipeline specifications

Software tools CLC Sequence Viewer, MEGA
Application Phylogenetics
Organisms Homo sapiens
Diseases Hepatitis C, Hepatitis C, Chronic
Chemicals Amino Acids