In this paper, we have presented a quantitative assay based on the TaqMan real-time PCR detection methodology for the detection of HBV DNA. Furthermore, we have demonstrated this detection system to be highly reproducible, with no statistically significant difference in variability, as well as to have the ability to detect HBV DNA between approximately 373 and 10 billion copies per ml without any further sample dilution or concentration. The low variability of this assay makes a twofold decrease or increase in the serum HBV DNA level already significant. The high reproducibility and linearity of the assay described proved to be comparable to those of two commercially available assays.
More than a decade has passed since the introduction and use of PCR technology for the detection of HBV DNA in serum. Although different commercial assays have become available enabling an easy-to-use platform, there is still progress to be made, since the detection range of these assays was shown to be limited. Only the Digene Hybrid Capture II microplate assay was able to detect HBV DNA over a wide range (8 × 103
to 1.7 × 109
copies per ml), although in the lower range a sample concentration step needs to be performed. The Roche HBV MONITOR assay is used for quantitative HBV detection in the lower range of HBV DNA detection in patients (400 to 4 × 107
copies/ml). The high variation of the HBV MONITOR assay within the lower detection range, which can even be more than 100%, is the main disadvantage of this assay (13
). The real-time PCR detection assay described here had a good correlation with both the Digene Hybrid Capture II microplate assay and the Roche HBV MONITOR assay and could be used for the monitoring of patients undergoing antiviral treatment. Regression analysis indicated that the standardization based on the VQC panel resulted in a large dynamic range and that data from all of the assays were interchangeable. Since it is usually not known in which range the HBV DNA level of a given sample is to be expected, especially when patients are receiving antiviral treatment, repeated testing is often necessary (13
). With the real-time detection assay described here, this problem is largely overcome. Furthermore, since the introduction of TaqMan technology, more cumbersome techniques for PCR quantitation seem to have become obsolete, due to the ability of this technology to quantify HBV DNA rather easily, without the need for the further PCR amplicon processing steps usually required to detect specific sequences (5
It has often been suggested that in-house PCR assays suffer from problems with standardization, false positivity, or contamination, making them unsuitable for routine clinical diagnostic use (14
). Quality control programs have indeed indicated that this is a problem (2
). It should, however, be realized that for both commercial and in-house assays, sample preparation is still the most difficult step to perform and to automate. Handling of samples and opening of tubes during sample preparation should be carried out under good laboratory practice principles in order to limit the problem of contamination. The complete abolishment of postamplification handling is an additional factor in limiting contamination. Our data further indicate that by using two independent isolates for one sample in our assay, the problem of false positivity is more easily observed and therefore controlled. Furthermore, participation in external quality control programs, as well as the continuous use of well-standardized validation panels, should further guarantee laboratory performance. Available standardized materials, like universal amplification mixtures, primers, and probes, for real-time PCR detection assays leave the input DNA as the only unknown factor. A further point of attention remains the possible inhibition of the amplification reaction by unknown components in the clinical sample. Inclusion of internal markers and selection of appropriate sample preparation devices and methods should limit this problem. We have, for instance, shown that certain sample preparation methods are better than others at removing the inhibitory effect of heparin (12
Collectively, our data show that HBV DNA quantitation with the real-time PCR detection technique resulted in a sensitive and highly reproducible assay, the results of which correlated well with those obtained with commercially available assays while offering the advantage of a wide dynamic range.