Optimization of Nested Polymerase Chain Reaction Conditions for Amplification of NS5B Gene Region of Hepatitis C Virus Genotype 6

Hepatitis C virus (HCV), a single stranded RNA virus, can be classified into six major genotypes. In Thailand, significant increase in HCV infections by genotype 6 has been reported. The goal of this study was to find suitable comditions for nested polymerase chain reaction (nested PCR) for the amplification of NS5B gene of HCV genotype 6. HCV-RNA was extracted from 28 serum samples with positive anti-HCV antibody. cDNA was synthesized and nested PCR was performed under five different conditions (I-V). Using protocols I-V, out of 28 samples, amplified products were detected in 11 (39.29%), 1 (3.57%), 2 (7.14%), 2 (7.14%), and 15 (53.57%), samples, respectively. The data indicated that protocol 5 could amplify and gave the highest yield for NS5B gene of HCV genotype 6. However, this condition could not be applied successfylly for all subtypes of HCV genotype 6. Thus further investigations need be performed by using new sets of primers under appropriately optimized conditions.