Rapid quantification of hepatitis B virus DNA by real-time PCR using fluorescent hybridization probes
Authors: Stephen K.N. Ho1, Wing-Cheong Yam1, Eric T.K. Leung1, Lei-Po Wong1, Jack K.H. Leung1, Kar-Neng Lai1, Tak-Mao Chan1
Affiliations: 1 Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China
Correspondence Tak-Mao Chan [email protected]
J. Med. Microbiol., May 2003 52: 397-402, doi: 10.1099/jmm.0.05071-0
Subject: Diagnostics, Typing And Identification
Received: 11/09/2002 Accepted: 20/01/2003 Published Online: 01/05/2003
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**1 to 10**8 copies per reaction (250–2.5 × 10**9 copies ml−1), with a rapid PCR cycling time of 35 min. The assay was validated with two EUROHEP HBV DNA standards (ad and ay 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 (P < 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−1), detectable only by the more sensitive LC-PCR assay. Levels of HBV DNA as measured by the two assays showed good correlation (r = 0.902; P < 0.001). The level of HBV DNA was significantly higher in HBeAg+ than anti-HBe+ samples (median 1.5 × 107 vs 4.6 × 104 copies ml−1; P < 0.01). It is concluded that this LC-PCR assay is clinically useful for the rapid, sensitive and accurate measurement of HBV DNA.
Abbreviations: CV, coefficient of variation; FRET, fluorescence resonance energy transfer; HBV, hepatitis B virus; HCII, Digene Hybrid Capture II; LC-PCR, LightCycler PCR.