High-Throughput Quantitative Analysis of Hepatitis B Virus DNA in Serum Using the TaqMan Fluorogenic Detection System

Abstract

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 branchedchain 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 bDNApositives were positive by TaqMan. Of the 77 samples with detectable HBV-DNA titers below 3.75 ؋ 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. Hepatitis B virus (HBV) infects approximately 400 million persons worldwide. HBV can cause pathology ranging from self-limited illness to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. 1 The progression of HBV disease is usually monitored by serologic assays. The branched-chain DNA assay (bDNA) (Chiron, Emeryville, CA) can be used to accurately determine viral load. However, the sensitivity of the bDNA test is limited to a range of approximately 6 ϫ 10 5 to 5 ϫ 10 9 HBV-DNA copies/mL. 2,3 This level of detection is far above what most authorities would consider optimal for suppression of HBV replication during therapy or for reducing transmission of HBV via sexual, bloodborne, or maternal-fetal routes. 4 Advances in antiviral therapy (lamivudine) and liver transplantation have also brought about a need for a more sensitive assay for HBV viral load. 5 We and others have used quantitative "real-time" fluorescent-based polymerase chain reaction (PCR) systems to detect nucleic acid from other viruses in plasma and to measure response to antiviral therapy. 6,7 In this article, we describe a new TaqMan-based quantitative PCR 8,9 for the detection of HBV DNA in serum, and compare this assay with the established serologic and bDNA tests for monitoring HBV infection.PCR primers (forward primer HBV1F: 5Ј CCG TCT GTG CCT TCT CAT CTG; reverse primer HBV1R: 5Ј AGT CCA AGA GTY CTC TTA TGY AAG ACC TT) and fluorescent probe (HBV1TAQ: 5Ј CCG TGT GCA CTT CGC TTC ACC TCT GC) were designed against a conserved region of the HBV genome overlapping the genes encoding the X-protein and DNA polymerase, defining an amplicon corresponding to bases 1549 to 1653 of the HBV genome. TaqMan PCR was performed as previously described. 6 To define the linear range of the assay, we used serially diluted bacterial plasmids encoding a wild subtype of HBV (ATCC #39629). The TaqMan PCR assay reproducibly quantitated bacterial plasmid samples ranging between 10 1 to 10 6 copies per reaction (Fig. 1). The standard deviation for each curve was less than 1% of each ...

Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B

“…20 The assay detects HBV-DNA using real-time fluorescent-probe PCR (TaqMan, Roche, Mannheim, Germany), with a lower limit of quantification of 100 copies/mL.…”

Quantitative measurement of hepatitis B virus DNA in different areas of hepatic lobules in patients with chronic hepatitis B

“…Real-time quantitative PCR was performed in a PE5700 real time quantitative PCR thermal cycler (Applied Biosystems, Foster City, CA), as described previously [Loeb et al, 2000, Chen et al, 2001. The amplification was performed in 50 ml of reaction mixture containing; 2Â Taqman Universal Master Mix (Roche Molecular Systems, Inc., Branchburg, NJ), 200 nM specific primers, 100 nM TaqMan probes, and 1 ml of extracted DNA solution.…”

“…The primers and probes were designed against a conserved region of the HBV genome overlapping the genes encoding the X-protein and DNA polymerase, defining an amplicon corresponding to bases 1549 to 1672 of the HBV genome, as described previously [Loeb et al, 2000]. Thermal cycling conditions were as follows: a hot start of 2 min at 508C and 10 min at 958C, followed by 40 cycles of 958C for 15 s, and 608C for 1 min.…”

Multiplex biomarker analysis biosensor for detection of hepatitis B virus

“…Currently, along with the development of biological techniques, molecular biology techniques based on gene tests have been established [3][4][5]. Among them, PCR is the main laboratory diagnostic test for the hepatitis B virus.…”