1887

Abstract

A highly sensitive and rapid assay has been developed to quantify hepatitis B virus (HBV) DNA, based on the fluorescence resonance energy transfer principle and real-time PCR, using the LightCycler and a pair of specific fluorescent hybridization probes. This LightCycler real-time PCR assay (LC-PCR) detected HBV DNA in a linear range from 10 to 10 copies per reaction (250–2.5 × 10 copies ml), with a rapid PCR cycling time of 35 min. The assay was validated with two EUROHEP HBV DNA standards ( and subtypes) and exhibited low intra-assay (< 6 %) and inter-assay (< 16 %) variation for both subtypes over the complete range of 7 orders of magnitude. The assay was evaluated clinically using serum samples from 120 HBsAg individuals and 45 healthy controls who were negative for both HBsAg and anti-HBc. Levels of HBV DNA were measured in these samples using both the LC-PCR and Digene Hybrid Capture II HBV DNA (HCII) assays. The prevalence rates for HBV DNA in the HBsAg serum samples were respectively 95 % (114/120) and 56 % (67/120) by LC-PCR and HCII ( < 0.01). All 67 HCII-positive samples tested positive with LC-PCR, while the 47 discordant samples showed low levels of HBV DNA (down to 265 copies ml), detectable only by the more sensitive LC-PCR assay. Levels of HBV DNA as measured by the two assays showed good correlation ( = 0.902; < 0.001). The level of HBV DNA was significantly higher in HBeAg than anti-HBe samples (median 1.5 × 10 vs 4.6 × 10 copies ml; < 0.01). It is concluded that this LC-PCR assay is clinically useful for the rapid, sensitive and accurate measurement of HBV DNA.

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2003-05-01
2020-08-05
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