Comparison of an In-house Qualitative Pcr Assay for Detection of Hepatitis B Virus Dna with Real Time Pcr Using Sybr® Green I

The HBV infection is a very serious health problem and there is need of sensitive and precise methods for diagnostics. The measurement of HBV DNA in serum with various molecular techniques has become an important part of the diagnostics procedure of this infection. In this study an in-house PCR method was compared with the Real Time PCR technique for detection of HBV DNA. It was founding in this case that they are both accurate but the Real Time technique has some important advantages. Introduction Hepatitis B virus (HBV) infection is a major health problem, with about 5% of the world’s population being chronically infected. Of these, about 1 million die each year due to progression to cirrhosis or hepatocellular carcinoma (1). HBV is the smallest DNA virus known, and its genome shows a highly compact organization. A unique aspect in the HBV replication cycle is that pregenomic mRNA serves as a template for synthesis of the first viral DNA strand by the reverse transcriptase (RT) polymerase of HBV (2). The RNAse H activity of the HBV DNA polymerase removes the mRNA during this process, and synthesis of the complementary second DNA strand is then started, generating a partially double-stranded DNA molecule for packaging in virions. When the virus enters the host, this molecule is extended into a fully double-stranded DNA molecule, thus starting a new replication cycle (3, 4, 5). HBV DNA can be detected in the blood in more than 90% of infected hosts who are positive for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). The use of polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the golden standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. The method utilizes a pair of synthetic oligonucleotides or primers, each hybridizing to one strand of a double-stranded DNA (dsDNA) target, with the pair spanning a region that will be exponentially reproduced. The hybridized primer acts as a substrate for a DNA polymerase (most commonly derived from the thermophilic bacterium Thermus aquaticus and called Taq), which creates a complementary strand via sequential addition of deoxynucleotides. The process can be summarized in three steps: (i) dsDNA separation at temperatures > 90°C, (ii) primer annealing at 50-75°C, and (iii) optimal extension at 72-78°C (6). Traditional detection of amplified DNA relies upon electrophoresis of the nucleic acids in the presence of ethidium bromide and visual or densitometric analysis of the resulting bands after irradiation by ultraviolet light (7). On the other hand, the Real Time PCR