HBV DNA Quantitation Using Real-time PCR

Jeong Heo; Won Ook Go; Gwang Ha Kim; Dae Hwan Kang; Geun Am Song; Mong Cho; Hyung Hoi Kim; Eeu Yup Lee

doi:10.3343/kjlm.2006.26.6.424

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Heo, Go, Kim, Kang, Song, Cho, Kim, and Lee: HBV DNA Quantitation Using Real-time PCR

Original Article

Korean J Leg Med 2006;26(6):424-430.

Published online: 10 February 2006

DOI: https://doi.org/10.3343/kjlm.2006.26.6.424

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, 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

³Department of Unit of Biomedical Informatics, Pusan National University, School of Medicine, Busan, Korea

Received 13 March 2006 Revised 31 October 2006 Accepted 12 November 2006

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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

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Fig. 1.

The genomic site (dotted box) of HBV DNA for primers and probes.

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.

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).

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.

Table 1.

Clinical and laboratory features of patients

Number of patients	52
Mean age (range), yr	43 (10–75)
Sex, male/female, n	34/18
Log₁₀ [Serum HBV DNA], mean±S.D, copies/mL	6.0±1.0
HBeAg positive/negative, n (%)	33/19 (63.5/36.5%)
Chronic hepatitis/liver cirrhosis, n	27/25

Table 2.

Primers of PCR amplication for standard plasmid

Primer

P1-Eco, forward, 5′-GAGAGAATTCTTTTTCACCTCTGCCTAATCA-3′

P2-Pst, reverse, 5′-GAGACTGCAGAAAAAGTTGCATGGTGCTGG-3′

Table 3.

Primers and probes of real-time PCR for HBV DNA quantitation

Primer

HBV Forward, 5′-GAC CAC CAA ATG CCC CTA T-3′

HBV 508 Reverse, 5′-AAG CGC TGC GTG TAG TTT CT-3′

Probe

HBV FL, 5′-GAV GCA GGW CCC CTA GAA GAA GAA-fluorescein-3′

HBV LC, 5′-LCRde-TCC CTC GCC TCG CAG ACG AAG TRC TS-phosphate-3′

Table 4.

Precision of real-time PCR on serial dilution of standard plasmid (5.0×10⁸ copies/mL)

Precision	Log₁₀ dilution	Observed (copies/mL)		CV (%)
Precision	Log₁₀ dilution	Mean	SD	CV (%)
Within-run	0	4.8×10⁸	5.6×10⁷	11.6
	−2	7.2×10⁶	2.6×10⁵	3.7
	−4	5.1×10⁴	3.8×10³	7.5
	−6	4.5×10²	1.1×10²	24.9
Between-day	0	4.7×10⁸	3.7×10⁷	7.8
	−2	5.9×10⁶	1.1×10⁵	18.6
	−4	5.5×10⁴	7.4×10³	13.6
	−6	5.4×10²	1.3×10²	24.7

Abbreviation: CV, Coefficient of variation.

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