PCR Amplification of Hepatitis B Virus in the Limiting Dilution Format

Hepatitis B virus has eight genotypes based on sequence heterogeneity and at least four HBV genotypes are present in Pakistan. If infected patient has mixed infection of different HBV genotypes, PCR amplification will result the product that may be hybrid of different HBV DNA molecules. Sequence analysis of PCR product amplified from such samples is often unreliable due to ambiguities in sequencing gel/electropherogram. In order to establish HBV DNA amplification from a single HBV molecule in a sample using limiting dilution format, two blood samples (NA004 and NA012) were collected from Lahore hospitals. Viral DNA was extracted from blood plasma and a regions of 1.1 Kb and 0.6 Kb were amplified by using two different sets of primers (NHBF1 & NHBR1 and B170AS & B2833S). Two limiting DNA dilutions, 1:256 and 1:4096 were found effective to get PCR product from a single HBV DNA molecule.