Premium
Real‐time PCR for detection and quantitation of hepatitis B virus DNA
Author(s) -
Chen Ren Wei,
Piiparinen Heli,
Seppänen Mikko,
Koskela Pentti,
Sarna Seppo,
Lappalainen Maija
Publication year - 2001
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.2027
Subject(s) - virology , taqman , real time polymerase chain reaction , hepatitis b virus , lamivudine , virus , hepatitis b , microbiology and biotechnology , dna , biology , medicine , gene , biochemistry , genetics
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 × 10 7 genome equivalents per ml. The detection limit for the real‐time PCR was 3.73 × 10 2 genome equivalents per ml (geq/ml), while it was 3.73 × 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. J. Med. Virol. 65:250–256, 2001. © 2001 Wiley‐Liss, Inc.