Rapid Quantification of Hepatitis B Virus DNA by Automated Sample Preparation and Real-Time PCR

Abstract

Monitoring of hepatitis B virus (HBV) DNA in serum by molecular methods has become the standard for assessment of the replicative activity of HBV. Several molecular assays for the detection and quantification of HBV DNA have been described. However, they usually lack automated sample preparation. Moreover, those assays, which are based on PCR, are limited by a short dynamic range (2 to 3 log units). In the present study, the use of RealArt HBV LC PCR Reagents in conjunction with automated extraction on the COBAS AMPLI-PREP analyzer was evaluated. Members of an HBV proficiency program panel were tested; linearity, interassay, and intra-assay variations were determined. The performance of the assay in a routine clinical laboratory was evaluated with a total of 117 clinical specimens. When members of the HBV proficiency program panel were tested by the new molecular assay, the results were found to be within ؎0.5 log unit of the results obtained by reference laboratories. Determination of linearity resulted in a quasilinear curve over more than 6 log units. The interassay variation of the RealArt HBV LC PCR Reagents by use of the automated sample preparation protocol ranged from 16 to 73%, and the intra-assay variation ranged from 9 to 40%. When clinical samples were tested by the new assay with the automated sample preparation protocol and the results were compared with those obtained by the COBAS AMPLICOR HBV MONITOR Test with manual sample preparation, the results for 76% of all samples with positive results by both tests were found to be within ؎0.5 log unit and the results for another 18% were found to be within between 0.5 and 1.0 log unit. In conclusion, the real-time PCR assay with automated sample preparation proved to be suitable for the routine molecular laboratory and required less hands-on time.Assays for quantification of hepatitis B virus (HBV) DNA in serum have been shown to be useful for pretreatment evaluation, clinical staging, monitoring of antiviral therapy, detection of the emergence of drug resistance, and detection of relapses after the discontinuation of antiviral therapy (1, 5).Several commercially available assays for quantification of HBV DNA have been brought onto the market and have been found to be useful for the routine diagnostic laboratory (2,6,8,9). Recently, real-time PCR has been introduced. This new technique combines amplification and detection of amplification products in the same closed vessel, thus reducing the analytical turnaround time as well as the risk of contamination (3, 7). To improve assay performance, ready-to-use reagents have been introduced (RealArt HBV LC PCR Reagents; Artus GmbH, Hamburg, Germany). They have been optimized for use on the LightCycler instrument (Roche Applied Science, Penzberg, Germany).For automated sample preparation, a new automated device, the COBAS AMPLIPREP analyzer (Roche Molecular Systems Inc., Branchburg, N.J.), has recently been developed (4, 10). Sample preparation protocols with this instrument were initially dependent on the use o...

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“…The interassay variation ranged from 6 to 42% and intra-assay variation ranged from 5 to 34%. These results correspond to those reported for other molecular assays based on PCR amplification (Kessler et al, 2000;Stelzl et al, 2004).…”

COBAS AmpliPrep-COBAS TaqMan Hepatitis B Virus (HBV) Test: a Novel Automated Real-Time PCR Assay for Quantification of HBV DNA in Plasma

“…Real-time PCR assays for HBV DNA based on the TaqMan technology, as well as on other real-time platforms, have been designed on manual, semiautomatic, or automatic nucleic acid extraction procedures suitable for small series. Thus far, these formats have shown an excellent correlation with conventional endpoint PCR systems, including CAHBM, which is the most commonly used assay in comparative evaluations (7,15,18,21,24,31,32).…”

“…The recently introduced real-time PCR technique represents the method of choice compared to previous, conventional endpoint PCR due to a very sensitive quantification of the viral load over a wide dynamic range (7,12,15,18,20,21,24,31,32,35,41). At present, sample preparation is a major weakness in molecular tests, and improvements are constantly introduced to decrease the variability of the techniques and the risk of contamination, such as ready-to-use reagents and automation of the extraction procedure.…”