Low copy number amplification of full-length hepatitis B virus gene and identification of in-vitro replication level in serum

Abstract:

Objective：To evaluate the replication of virus by extracting virus DNA from low copy number of hepatitis B virus （HBV） serum samples，doing PCR amplification to acquire full-length HBV DNA fragments and transfecting HuH7 cells with full-length HBV DNA fragments after treatment. Methods：Primers for fragment and full-length amplification containing BspQⅠ restrictive en－zyme cutting site were designed and HBV DNA from patients was used as templates. Full-length HBV was acquired by nested PCR combined with sectional amplification. Closed circular DNA was got through restrictive enzyme digestion and T4 DNA ligase ligation. HBV replication level was analyzed by Southern blot after HuH 7 cells being transfected with closed circular HBV DNA. Results：Five complete HBV genomes were successfully obtained using the new PCR method. After digestion，ligation and tansfection experiments，Southern blot results all supported replication expression. Conclusions：This method lays a foundation for investigating the correlation of different HBV replication levels and DNA variation in patients with low copy number of virus.