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Diagnostic Immunology



Korean J Lab Med. 2006 Dec;26(6):424-430. Korean.
Published online December 20, 2006.  https://doi.org/10.3343/kjlm.2006.26.6.424
Copyright © 2006 The Korean Society for Laboratory Medicine
HBV DNA Quantitation Using Real-time PCR
Jeong Heo, M.D.,1 Won Ook Go, M.D.,1 Gwang Ha Kim, M.D.,1 Dae Hwan Kang, M.D.,1 Geun Am Song, M.D.,1 Mong Cho, M.D.,1 Hyung Hoi Kim, M.D.,2,3 and Eeu Yup Lee, M.D.2
1Department of Internal Medicine, Pusan National University, School of Medicine, Busan, Korea.
2Department of Laboratory Medicine, Pusan National University, School of Medicine, Busan, Korea.
3Unit of Biomedical Informatics, Pusan National University, School of Medicine, Busan, Korea.
Received March 13, 2006; Accepted November 12, 2006.

Abstract

Background

Accurate measurement of the concentration of hepatitis B virus (HBV) DNA in clinical samples is important for the appropriate treatment of patients and evaluation of their therapeutic responses. In addition, the concentration of HBV DNA in the serum of patients with chronic HBV infection has a very broad range. Real-time PCR is very sensitive and useful to detect HBV DNA in a wide range of concentrations. We designed new primers and probes for real-time PCR to detect HBV DNA.

Methods

Primers and probes specific for HBV were designed. EUROHEP HBV DNA standards (NIBSC, Hertfordshire, UK) with the HBV DNA concentration of 7.0×104 copies/mL was used to determine the calibration curve and efficacy for the real-time PCR assay. Sensitivity, dynamic range, and precision were evaluated. The correlation between the real-time PCR and Cobas Amplicor HBV Monitor™ assay in the measurement of serum HBV DNA concentrations in 52 patients with chronic HBV infection was evaluated.

Results

The sensitivity of the assay was approximately 6.08×102 copies/mL for HBV, and the quantitation was accurate and reproducible over a wide dynamic range from 6.1×02 to 6.5×109 copies/mL without any dilution of specimens. The assay showed low coefficients of variation of repeatability (3.7-24.9%) and reproducibility (7.8-24.7%). The results were found to correlate well with those obtained by Cobas Amplicor HBV Monitor™ kit.

Conclusions

We provide a new in-house method for the measurement of serum HBV DNA using real-time PCR, which enables us to detect HBV DNA rapidly, sensitively, and accurately.

Keywords: HBV DNA quantitation; Real time PCR; Cobas amplicor

Figures


Fig. 1
The genomic site (dotted box) of HBV DNA for primers and probes.
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Fig. 2
Sigmoid fluorescence curves obtained with serial dilutions of standard plasmid. A, distilled water; B, 5.0×109 copies/mL; C, 5.0×107 copies/mL; D, 5.0×105 copies/mL; and E, 5.0×103 copies/mL.
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Fig. 3
The dilutional linearity of HBV DNA quantitation using real-time PCR. The standard curve was obtained with serial dilutions of standard plasmid (♦, mean of 5 experiments).
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Fig. 4
Correlation between real-time PCR HBV DNA quantitation and the Cobas Amplicor HBV Monitor™, test (P<0.001). Regression analysis of 52 sera of chronic hepatitis B patients analysed by both test methods. The real-time PCR HBV DNA quantitation test was duplicated. All specimens had titers above 1,000 copies/mL. Samples above 2.0×105 copies/mL in the the Cobas Amplicor HBV Monitor™ test were diluted.
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Tables


Table 1
Clinical and laboratory features of patients
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Table 2
Primers of PCR amplication for standard plasmid
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Table 3
Primers and probes of real-time PCR for HBV DNA quantitation
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Table 4
Precision of real-time PCR on serial dilution of standard plasmid (5.0×108 copies/mL)
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