Quantitation of hepatitis B virus genomic DNA by real-time detection PCR

Abstract

Quantitation of hepatitis B virus (HBV) DNA in serum is a useful method for the monitoring of HBV replication. We attempted to develop a quantitative assay system for HBV DNA that is more sensitive, accurate, and reproducible than existing systems. We detected HBV DNA by real-time detection PCR (RTD-PCR) based on Taq Man chemistry. The efficacy of this assay was evaluated by quantitatively measuring sequential levels of synthetic DNA and DNA in clinical serum samples. The detection limit of this system was as few as 10 DNA copies/reaction. A linear standard curve was obtained between 10(1) and 10(8) DNA copies/reaction. The coefficient of variation for both intra- and interexperimental variability indicated remarkable reproducibility. This system detected HBV DNA in 100% of chronic hepatitis B patients tested and never detected HBV DNA in healthy volunteers who were negative for HBV markers. These observations suggest that RTD-PCR is an excellent candidate for a standard HBV quantification method.

FIG. 1

Clinical course of a case of acute exacerbation in an HBV carrier. The dotted area indicates HBV DNA levels below the detection limit of the bDNA assay.

FIG. 2

Amplification plots and standard curves of synthetic HBV DNA based on three sets of primers and probe. Set 1 (a and d) and set 2 (b and e) were located in the S region, and set 3 (c and f) was located in the X region. Serial 10-fold dilutions of standard HBV DNA from 10¹ to 10⁸ copies/reaction tube were prepared. (a to c) ΔRn was plotted against each cycle number. ΔRn increases during PCR as the amplicon copy number increases until the reaction reaches a plateau. The number of copies of each standard HBV DNA are shown. (d to f) Ct was plotted against each copy number. Ct represents the PCR cycle at which reporter signal can first be detected.

FIG. 3

The correlation between serum HBV DNA levels determined by RTD-PCR and those determined by bDNA assay was studied with samples collected from 46 patients with chronic hepatitis B. (a and c) Correlation between serum HBV DNA levels in 25 of 46 patients determined by bDNA assay and RTD-PCR using set 1 and set 3 primers and probes. (b) Correlation between serum HBV DNA levels determined by bDNA assay and RTD-PCR using set 2 primers and probe. (d) Correlation between serum HBV DNA levels determined by RTD-PCR using primers and probes located in the S region (set 1) and the X region (set 3). Correlation coefficients are indicated in each panel. Broken lines indicate the detection limit of the bDNA assay.