Real-time PCR for detection and quantitation of hepatitis B virus DNA.
A sensitive and reproducible real-time PCR assay based on TaqMan technology was developed for the detection and quantitation of hepatitis B virus (HBV) DNA in serum, and compared with an "in-house" qualitative PCR assay. HBV DNA was measured in 125 serum samples from 76 hepatitis B patients, consisting of 22 patients with an acute infection, 20 patients with a previous history of hepatitis B infection, and 34 patients with a chronic hepatitis B. Four patients with a chronic infection were treated with either an IFN-alpha monotherapy or a combination of IFN-alpha and lamivudine. Twenty-nine sera from healthy individuals and non-hepatitis B patients served as negative controls. The assay was validated by using a 10-fold dilution series of the World Virological Quality Control (VQC) sample containing 3.73 x 10(7) genome equivalents per ml. The detection limit for the real-time PCR was 3.73 x 10(2) genome equivalents per ml (geq/ml), while it was 3.73 x 10(3) geq/ml for the in-house PCR. The real-time PCR assay had an 8-logarithm dynamic range spanning from 10(2) to 10(10) geq/ml. In clinical serum samples, the real-time PCR and the in-house PCR detected HBV DNA in 81% (101/125) and 66% (83/125) of samples, respectively. HBV DNA was not detected among the negative controls by either of these assays. In conclusion, real-time PCR is a sensitive, specific, and a reproducible approach for the detection and quantitation of HBV DNA in clinical serum samples, useful also for monitoring the efficacy of antiviral treatment.
Chen
R W
RW
Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland.
Piiparinen
H
H
Seppänen
M
M
Koskela
P
P
Sarna
S
S
Lappalainen
M
M
D000208:Acute Disease; D000998:Antiviral Agents / Q000627:therapeutic use; D003201:Computers; D004279:DNA, Viral / Q000097:blood*; D004359:Drug Therapy, Combination; D005500:Follow-Up Studies; D006509:Hepatitis B / Q000097:blood / Q000188:drug therapy / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D019694:Hepatitis B, Chronic / Q000821:virology; D006801:Humans; D016898:Interferon-alpha / Q000627:therapeutic use; D019259:Lamivudine / Q000627:therapeutic use; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity
D000998:Antiviral Agents; D004279:DNA, Viral; D016898:Interferon-alpha; D019259:Lamivudine