Background and Aim: Using sensitive and specific laboratory techniques such as PCR is necessary on diagnosis of hepatitis which caused by hepatitis B virus (HBV). Common serological methods didn’t have the capability of quick and accurate detection of infection detection thought molecular methods such as PCR are effective tools for evaluation prevalence of HBV. A different result for the reason of not being standard is one of the disadvantages of this rich molecular technique. One of the most significant preventives in widespread usage of diagnostic PCR is not having proper PCR internal controls. The aim of this study is designing and constructing plasmid internal control (IAC) for identification of PCR inhibitors and afterward usage in diagnostic laboratories.

Materials and methods: In this study for constructing the internal control, PCR specific primers for HBsAg gene of hepatitis B virus were optimized then sensitivity and specificity were determined. Also Composite Primers for HBV-IAC were designed and amplified by PCR then cloned. Minimum number of IC in each PCR reaction was examined through dilution and PCR results with IC.

Result: By using special primers HBV PCR product was 262bp and IAC-HBV product was 660bp which had desirable different in sizes. Minimum number of IC in each reaction was 1000. Maximum of PCR sensitivity with IC for hepatitis B virus DNA was determined as 16 million Particles. In specificity test with different factors no non specific product was observed.

Conclusion: For Molecular detection of HBV using an internal control can recognize the errors and will standardize this sensitive technique.