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Abstract
Background
Hepatitis B virus (HBV) DNA quantification is important for the management of HBV infection and identification of the development of resistance. The susceptibility to contamination and more variable reproducibility of results with the conventional HBV DNA quantification method have raised the need of a more simple and accurate method for HBV DNA quantification. Real-time quantitative PCR assays recently introduced in the laboratory can meet these needs. In this study, we evaluated the performance of the Real-Q HBV Quantification kit developed in Korea.
Methods
We evaluated the recovery of DNA extraction, the interference of internal control, an analytical sensitivity, specificity, and reproducibility, a clinical specificity, and a reportable range of the Real-Q HBV Quantification kit. The quantification result was also compared to that obtained by the Digene Hybrid-Capture II.
Results
The mean percent recovery was 108.6% and there was no interference with the internal control on DNA extraction. None of HIV, hepatitis C virus, or cytomegalovirus showed a cross-reactivity with HBV. This assay detected HBV DNA in a linear range from 102 to 1010 copies/mL, with the detection limit of 56 copies/mL. The assay exhibited a low within-run CV (coefficient of variation) (8.7–11.9%), between-run CV (10.5–14.7%), and between-day CV (13.2–21.4%). No HBV DNA was detected in any of 100 samples without HBV, resulting in a clinical specificity of 100%. The levels of HBV DNA showed a good correlation with those determined with Digene Hybrid-Capture II (R2=0.9827).
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Fig. 1.
Quantitative correlation between reference from HBV viral DNA assay validation kit (AcroMetrix corporation) and observed value by Real-Q HBV Quantification kit (BioSewoom Inc.).
Fig. 2.
Linearity range of Real-Q HBV Quantification kit (BioSewoom Inc.). The straight line was determined by a linear regression of the log 10 estimated concentrations with the log 11 nominal concentrations. Linearity was found from 102 to 1010 copies/mL.
Fig. 3.
Quantitative comparison of Real-Q HBV Quantification kit (BioSewoom Inc.) with Digene Hybrid-Capture II (Murex Diagnostics).
Table 1.
Detection limit of Real-Q HBV Quantification kit
| Input HBV (copies/mL) |
Result |
||
|---|---|---|---|
| Replicates | Positive reaction | % | |
| 225 | 50 | 50 | 100 |
| 112.5 | 50 | 50 | 100 |
| 84 | 50 | 49 | 98 |
| 56 | 50 | 48 | 96 |
| 28 | 50 | 39 | 88 |
Table 2.
Analytical specificity of Real-Q HBV Quantification kit
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