Korean J Lab Med.  2006 Dec;26(6):424-430. 10.3343/kjlm.2006.26.6.424.

HBV DNA Quantitation Using Real-time PCR

• Heo J
• Go WO
• Kim GH
• Kang DH
• Song GA
• Cho M
• Kim HH
• Lee EY

• KMID: 1889821
• DOI: http://doi.org/10.3343/kjlm.2006.26.6.424

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 x 10(4) 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(TM) 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 x 10(2) copies/mL for HBV, and the quantitation was accurate and reproducible over a wide dynamic range from 6.1 x 0(2) to 6.5 x 10(9) 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(TM) 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.

Keyword

HBV DNA quantitation; Real time PCR; Cobas amplicor

MeSH Terms

Calibration
DNA*
Hepatitis B virus
Humans
Real-Time Polymerase Chain Reaction*
DNA

Figure

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  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 realtime 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.

Reference

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