A competitor DNA template for the molecular quantification of the hepatitis B virus.

The molecular determination of viral load in the serum represents the most valuable prognostic marker of HBV infection. In this paper, a new molecular assay for the quantitative measurement of HBV presence is described. It is based on PCR performing with a HBV-specific competitor DNA template. For the construction of the DNA template, a HBV DNA-originated 436 bp DNA fragment was modified by introducing a 110 bp deletion and cloned into pUC19. The resulting vector serves as the competitor DNA template in the competitive PCR. Post-PCR, the competitor DNA generates an amplified fragment of 306 bp; it could be easily distinguished from the product generated from the viral-originated DNA product (416 bp) when the same primers are used. The quantitative ratio between the two products enables the quantitative determination of viral load. The range of the HB-PCR assay is from 3 x 10(4)to 6 x 10(10) particles/ml. A serum HBV load determination performed by HB-PCR assay indicated a close correlation with the results of the Quantiplex HBV DNA assay (bDNA). The HB-PCR assay is cheap, reliable and easy to use in any laboratory working with PCR methods.