Rapid quantification of hepatitis B virus DNA by real-time PCR using fluorescent hybridization probes

• DOI: 10.1099/jmm.0.05071-0
• Publication Date: 2003-04-29T15:13:50Z
• Authors: Stephen K.N. Ho; Wing-Cheong Yam; Eric T.K. Leung; Lei-Po Wong; Jack K.H. Leung; Kar-Neng Lai; Tak-Mao Chan
• Keywords: 0301 basic medicine; 570; Hepatitis B virus; Time Factors; Carrier State - diagnosis - virology; Hepatitis B e Antigens - blood; Polymerase Chain Reaction; Sensitivity and Specificity; Fluorescence; 03 medical and health sciences; Viral - blood; Hepatitis B virus - genetics - isolation & purification; Humans; Hepatitis B e Antigens; Hepatitis B Surface Antigens - blood; Polymerase Chain Reaction - methods; In Situ Hybridization; In Situ Hybridization, Fluorescence; 0303 health sciences; Hepatitis B Surface Antigens; Hepatitis B - diagnosis - virology; Reproducibility of Results; DNA; Viral Load; Hepatitis B; 3. Good health; Case-Control Studies; Carrier State; DNA, Viral; DNA Probes - diagnostic use; DNA Probes; DNA, Viral - blood

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 x 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 x 10(7) vs 4.6 x 10(4) 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.