Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation.

ABSTRACT: To develop a Fok-I nested polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) method for the detection of hepatitis B virus X region (HBx) V5M mutation.Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma (HCC) and 77 carrier patients]. To identify V5M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I (GGA TGN9?) was done. For size comparison, the enzyme-treated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.The assay enabled the identification of 69 patients (sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5M prevalence in HCC patients was significantly higher than in carrier patients (47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBxAg V5M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBxAg V5M mutation in chronic patients with genotype C2 infection.

SUBMITTER: Kim H 

PROVIDER: S-EPMC4679770 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature