High-throughput quantitative analysis of hepatitis B virus DNA in serum using the TaqMan fluorogenic detection system.
Reproducible quantitative assays to detect viral nucleic acids have proven useful in defining disease progression and following response to therapy in a wide variety of viral infections. We describe the development of a quantitative assay to detect hepatitis B virus (HBV) DNA using real-time fluorescent-probe polymerase chain reaction (PCR) (TaqMan). The assay is highly reproducible, highly automated, and much more sensitive than the currently used branched-chain DNA (bDNA) assay for HBV. The quantitative PCR assay accurately detected samples ranging from 10 to 10(9) copies of HBV DNA per milliliter. Of 157 serum samples submitted for HBV quantitation, 119 were positive by TaqMan PCR versus only 55 by bDNA (P <.001). All 55 bDNA-positives were positive by TaqMan. Of the 77 samples with detectable HBV-DNA titers below 3.75 x 10(5) copies by TaqMan, only 13 were detected by bDNA. We tested 119 patients negative for all HBV serologic markers, and all tested negative in the TaqMan assay. HBV DNA was detected by TaqMan in 164 of 195 (84%) of hepatitis B surface antigen (HBsAg)-positive samples. Among hepatitis B e antigen (HBeAg)-positive samples, median titers were 4. 3 x 10(6) copies/mL versus 322 copies/mL in HBeAg-negative samples (P =.012). The TaqMan assay for HBV DNA is highly sensitive and reproducible and thus appears useful in accurately defining levels of viral replication among persons with HBV infection.
Loeb
K R
KR
Department of Laboratory Medicine, University of Washington, Seattle 98109-1024, USA.
Jerome
K R
KR
Goddard
J
J
Huang
M
M
Cent
A
A
Corey
L
L
D003199:Computer Systems; D004247:DNA / Q000737:chemistry; D004279:DNA, Viral / Q000097:blood*; D005456:Fluorescent Dyes; D006514:Hepatitis B Surface Antigens / Q000032:analysis; D006513:Hepatitis B e Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000097:blood*
D004279:DNA, Viral; D005456:Fluorescent Dyes; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D004247:DNA; D019914:Taq Polymerase