Rapid quantification of hepatitis B virus DNA by real-time PCR using fluorescent hybridization probes.
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.
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
Stephen K N
SKN
Ho
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
Wing-Cheong
WC
Yam
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
Eric T K
ETK
Leung
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
Lei-Po
LP
Wong
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
Jack K H
JKH
Leung
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
Kar-Neng
KN
Lai
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
Tak-Mao
TM
Chan
D002353:Carrier State / Q000175:diagnosis* / Q000821:virology; D016022:Case-Control Studies; D015342:DNA Probes; D004279:DNA, Viral / Q000097:blood*; D006509:Hepatitis B / Q000175:diagnosis* / Q000821:virology; D006514:Hepatitis B Surface Antigens / Q000097:blood; D006513:Hepatitis B e Antigens / Q000097:blood; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D019562:Viral Load
D015342:DNA Probes; D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens