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.
Ho
Stephen K N
SKN
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
Yam
Wing-Cheong
WC
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
Leung
Eric T K
ETK
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
Wong
Lei-Po
LP
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
Leung
Jack K H
JKH
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
Lai
Kar-Neng
KN
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
Chan
Tak-Mao
TM
Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China.
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