The most conserved regions for 15 hepatitis B virus complete genome of different subtypes were aligned using PCGENE software CLUSTAL to design a new pair of primer that can bind to each subtype of hepatitis B virus (HBV), to amplify PreS region of HBV genome. A pair of primer from these conserved patches was selected using software PRIMER and named as Nhepf1 and Nhepr1. Nhepf1, forward primer bound 2362-2385 nucleotides and Nhepr1, reverse primer bound 260-283 nucleotide amplify 1.12 Kb region of HBV genome that contain PreS sequence. The pair of primer was optimized for PCR. Nhepf1 and Nhepr1 annealed well at 50°C to subtype adw2 (American), adr4 (Japanese) and Pakistanian patient derived HBV DNA without any nonspecific bands. The results were found to be highly reproducible with greater accuracy.
Key words: Hepatitis B virus (HBV) genome, HBV conserved region, polymerase chains reaction (PCR) primers, primer designing.