Real-time PCR assay using molecular beacon for quantitation of hepatitis B virus DNA.
Levels of hepatitis B virus (HBV) DNA in the blood serve as an important marker in monitoring the disease progression and treatment efficacy of chronic HBV infection. Several commercial assays are available for accurate measurement of HBV genomic DNA, but many of them are hampered by relatively low sensitivity and limited dynamic range. The aim of this study was to develop a sensitive and accurate assay for measuring HBV genomic DNA using real-time PCR with a molecular beacon (HBV beacon assay). The performance of this assay was validated by testing serial dilutions of the two EUROHEP HBV DNA standards (ad and ay subtypes) of known concentrations. The assay showed low intra-assay (<7%) and interassay (<5%) variations for both subtypes. Its dynamic range was found to be 10(1) to 10(7) copies per reaction (1.0 x 10(2) to 1.0 x 10(9) copies ml(-1)). The assay was further evaluated clinically using serum samples from 175 individuals with chronic hepatitis B. The HBV DNA level measured by this assay showed good correlation with that measured by the commercially available COBAS AMPLICOR HBV Monitor test (r = 0.901; P < 0.001). The higher sensitivity and broader dynamic range of this assay compared to the existing commercial assays will provide an ideal tool for monitoring disease progression and treatment efficacy in HBV-infected patients, in particular for those with low levels of HBV viremia.
Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong.
Simon Siu-Man
SS
Sum
Danny Ka-Ho
DK
Wong
Man-Fung
MF
Yuen
He-Jun
HJ
Yuan
Jian
J
Yu
Ching-Lung
CL
Lai
David
D
Ho
Linqi
L
Zhang
D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D019694:Hepatitis B, Chronic / Q000097:blood / Q000175:diagnosis*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014162:Transfection
D017931:DNA Primers