A rapid real-time quantitative polymerase chain reaction for hepatitis B virus

Journal of Virological Methods 93 (2001) 105– 113 www.elsevier.com/locate/jviromet

A rapid real-time quantitative polymerase chain reaction for hepatitis B virus K. Brechtbuehl a, S.A. Whalley b, G.M. Dusheiko b, N.A. Saunders a,* a

Molecular Biology Unit, Sexually Transmitted and Blood Borne Virus Laboratory, Central Public Health Laboratory, 61 Colindale A6enue, London NW 9 5HT, UK b Department of Medicine, Royal Free and Uni6ersity College Medical School, London, UK Received 5 October 2000; received in revised form 4 January 2001; accepted 9 January 2001

Abstract Quantification of hepatitis B virus (HBV) DNA in serum is important for monitoring treatment. A rapid and cost effective alternative to the methods available currently was developed based on a real-time quantitative polymerase chain reaction (PCR) done in the LightCycler™ apparatus. Primers and a probe for sequences of the surface gene of HBV were designed and quantification achieved by reference to standards containing known concentrations of the target sequence. A single copy of the HBV genome could be detected if present in the reaction mixture. The quantitative range of the assay was from 4 ×102 to 1.3×1010 surface gene copies/ml serum. Nested PCR was required for quantification in the lower part of this range ( B 105 copies). The real-time PCR and Amplicor Monitor (Roche) tests performed comparably at virus concentrations below 106 copies/ml. The commercial test underestimated higher concentrations of virus. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Hepatitis B virus; Quantification; Real-time; Polymerase chain reaction

1. Introduction Infection with the hepatitis B virus (HBV), especially in infancy, is a common cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma (Beasley, 1988). Although effective neonatal immunisation is now available it is estimated that the number of HBV carriers worldwide still * Corresponding author. Tel.: + 44-20-82004400, ext.: 3070; fax: + 44-20-82001569. E-mail address: [email protected] (N.A. Saunders).

stands at 400 million worldwide and is set to rise. Some carriers eventually clear virus markers from the blood, but many succumb to one of the serious diseases that result from HBV infection. Treatment of carriers to reduce the level of viral replication can be expected to decrease morbidity and the potential for onward transmission of infection (Perrillo, 1991). Treatment for HBV infection currently involves drugs such as interferon a-2b (Wong et al., 1993) and lamivudine (Dienstag et al., 1995), and monitoring of HBV concentration in plasma is consid-

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ered essential for the successful management of patients on drug therapy. Increases in the virus concentration may also give an early indication of the emergence of drug resistant variants (Lai et al., 1998). Measurement of serum HBV DNA can be used to assess the infection risk posed by HBV carriers who perform invasive surgery. Commercial methods available currently for measurement of HBV concentration in plasma depend upon direct hybridisation (Hendricks et al., 1995) or competitive polymerase chain reaction (PCR) (Kessler et al., 1998; Noborg et al., 1999). A rapid and sensitive alternative method based on real-time PCR in the LightCycler™ apparatus is described.

2. Materials and methods

2.1. The principle of the HBV real-time PCR The quantifying reaction described below takes place in glass capillaries within the LightCycler™ real-time PCR apparatus (Idaho Technology, Idaho Falls, UT). Hybridisation of a probe to its target sequence results in an increase in light emitted from the Cy5 fluor, which is attached to the probe. This increase, which is measured in the LightCycler™ during the temperature cycling process, is due to fluorescent resonance energy transfer between SYBR-green I and Cy5. SYBR-green, a dye which binds double stranded DNA, and Cy5 are brought into proximity when the probe hybridises to its target sequence resulting in an increase in the efficiency of fluorescent resonance energy transfer. This system is known as Bi-probe and is patented by the Defence Evaluation and Research Agency and Bio/Gene Ltd., Kimbolton (Great Britain patent GB2333359A, 21 July 1999). During the temperature cycle optimal levels of probe binding were assumed to occur after the 2 s hold at 58°C. The LightCycler™ then illuminates the contents of each capillary at the excitation wavelength of SYBR-green I. Photons emitted in the emission peak of the Cy5 fluor (channel F2, 675 nm) and in the emission peak of SYBR-green I (channel

F1, 540 nm) are monitored simultaneously. The number of cycles required to produce a signal that is significantly above background is inversely related to the log10 input copy number. Quantitative values for samples can therefore be estimated from standard curves prepared using standards of known viral concentration. Following hybridisation the temperature within the sealed reaction capillary is increased progressively with constant monitoring of the level of fluorescence in both channels. Close to the melting temperature of the probe the equilibrium between bound and unbound probe shifts towards the disassociated state and a decrease in fluorescence occurs. When the temperature and fluorescence data are plotted as a first-derivative (− dF/dT versus T) a peak is generated at the melting temperature. The temperature at which the melting peak occurs is characteristic of the probe/target sequence duplex. Samples and standards are considered to have given a positive PCR result if they give a melting peak in the range 60–70°C.

2.2. Sample preparation Archived serum samples from 81 HBV surface antigen positive blood donors collected in the United States of America and standard sera (Centraal Laboratorium van de Bloedtransfusiedienst, Amsterdam) were analysed. DNA was extracted using the method of Boom et al. (1991) using L6 and L2 buffers and silica suspension (Severn Biotech, Stourbridge). Briefly, serum or plasma (150 ml) was mixed with 1 ml of L6 buffer and 20 ml of silica suspension to disrupt the viral particles and allow the released nucleic acid to bind to the solid phase. The silica was then washed sequentially with 1 ml L2 buffer (twice), 1 ml 70% ethanol (twice) and 1 ml acetone then dried. Bound nucleic was eluted from the silica into 20 ml of TE buffer containing 10 mg/ml proteinase K (Life Technologies, Glasgow) and 5 mg/ml Herring sperm DNA (Sigma, Poole) by digestion at 56°C for 10 min. Silica particles were removed by brief centrifugation. DNA extracts were stored at − 20°C.

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2.3. Probes and primers The probes and primers for this study were designed with reference to alignments of the surface gene of HBV and are shown in Table 1. All oligonucleotides were PSF® grade and were synthesised and supplied by MWG-Biotech UK Ltd., Milton Keynes. The probes were RP-HPLC purified and tagged with Cy5 and biotin at their 5% and 3% ends, respectively. Probes and primers were diluted to 100 pmol/ml (oligonucleotides supplied at a concentration of B 100 pmol/ml were stored as supplied) in sterile nuclease free water for long-term storage at − 20°C.

2.4. Preparation of quantitati6e standards PCR products for cloning were prepared using DNA extracted from the serum of an acute case of HBV (genotype D) infection. The amplification conditions were as described below (PCR section) for a single round PCR in the LightCycler™ except that the melting peak determination was omitted and replaced by a 10 min hold at 74°C. For the outer surface primer pair this procedure gave a single band of the expected size (281 bp) after agarose gel electrophoresis and ethidium bromide staining. A 1 ml aliquot of the PCR product was used directly for cloning using the TOPO cloning kit (Invitrogen, Leek, The Netherlands). Clones were screened by PCR using conditions described below (PCR section) except that 1 Table 1 Probes and primers used for the HBV surface gene quantitative real-time PCR Outer plus Outer minus Inner plus Inner minus Probe

5%-GAT GTG TCT GCG GCG TTT TA-3% bases 376–395a 5%-CTG AGG CCC ACT CCC ATA GG -3% bases 637–656 5%-GTG TCT GCG GCG TTT TAT CA-3% bases 379–398 5%-AGA GGA CAA ACG GGC AAC AT-3% bases 461–480 5%-Cy5 CCT GCT GCT ATG CCT CAT CTT C Biotin-3% bases 412 –433

a Numbers of bases refer to EMBL accession D28880 (strain Fukuoka Red Cross HBV e-negative 1992).

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ml dilute cell suspension was substituted for DNA extract. Plasmids were purified from cells grown overnight in Luria-Bertani medium using a miniprep kit (Sigma). Plasmid preparations were quantified spectrophometrically and standards were prepared by serial dilution in a solution of 5 mg/ml herring sperm DNA. 2.5. Polymerase chain reaction The potential for contamination of PCR reactions was minimised by using separate rooms for handling reagents, samples and PCR reaction mixtures. The first round of PCR amplification was performed on a standard Perkin –Elmer 9600 cycles (Perkin Elmer, Warrington) in 0.2 ml thin walled polypropylene tubes. The amplification mixture, in a final volume of 50 ml, contained 50 mM Tris –HCl pH 8.3, bovine serum albumin (0.5 mg/ml), 3 mM MgCl2 200 mM dNTPs and 2 units Platinum Taq polymerase (Life Technologies, Paisley) 5 pmol of each surface gene outer primer and 5 ml of serum extract or plasmid standard. The thermal programme comprised an initial denaturation at 94°C for 30 s followed by 15 amplification cycles each of three steps: 94°C hold for 20 s, 50°C for 30 s and 74°C for 30 s. Maximum ramp rates were used throughout. PCR reactions in the LightCycler™ were identical whether they were the second round giving the nested reaction or the single round PCR. Each reaction in a final volume of 10 ml contained 50 mM Tris –HCl pH 8.3, bovine serum albumin (0.5 mg/ml), 3 mM MgCl2, 200 mM dNTPs, 0.4 units Platinum Taq polymerase (Life Technologies), 5 pmol of the inner primer pair, 2.5 pmol surface probe, SYBR-green I 1:10 000 (Bio/Gene) and 1 ml of HBV extract. Samples for the nested PCR were 1 ml of a 10-fold dilution of the first round PCR products derived from either serum extracts or plasmid standards. The thermal programme comprised an initial denaturation at 94°C for 20 s followed by 40 amplification cycles each of four steps: 92°C held for 0 s, 55°C for 0 s, 58°C for 2 s and 74°C for 5 s. Fluorescence was measured once in each cycle after the 58°C hold. Maximum ramp rates were used (up to 20°C/s) except for the transition from 55 to 58°C when the ramp rate was 3°C/s.

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Fig. 1. Real-time PCR – the relationship between crossing threshold cycle and concentration of HBV DNA in the target. A typical plot is shown for standards containing from 5 × 101 to 107 copies of the PCR target (y axis) against the crossing threshold cycle number (x axis).

The melting characteristics of the PCR products and of the probe/PCR product duplex were determined in the LightCycler™ following amplification. The tube was heated to 92°C to melt all dsDNA present then cooled to 50°C at maximum ramp rate allowing a proportion of the PCR products to reanneal and probe to anneal to its target site on the ssDNA. The temperature was raised to 90°C at a rate of 0.2°C/s with continuous fluorescence acquisition (in continuous fluorescence acquisition mode the LightCycler™ continually cycles through the individual samples measuring fluorescence and temperature concurrently). PCR using the quantitative Roche monitor kit was carried out according to the manufacturers’ instructions.

The single round PCR included external standards at 0, 50, 102, 103, 105 and 107 plasmid copies. Each nested quantitative PCR run included external standards containing 0, 25, 50, 5× 102, 5× 103 or 5×105 copies of the surface gene target. Quantification values below 102 copies in the single round PCR (equating to 1.3× 104 viral genomes/ml serum) or 15 copies in the nested PCR (equating to 4× 102 viral genomes/ml serum) were below the cut-off values selected for these assays. All samples were first tested in the single round PCR. If values below the cut-off were obtained, the samples were retested in the nested PCR.

3. Results

2.6. Quantification

3.1. Sensiti6ity

The threshold value of PCR product accumulation (detection channel F2) used in constructing of the standard curve (Fig. 1) was set so that the data points for the quantitative standards gave the best fit to a straight line (log input copy number versus cycle number at threshold crossing). Numerical values for viral load in each sample were calculated using the LightCycler™ software.

The sensitivity of the LightCycler™ assays using a single round of amplification was between 10 and 50 copies of the HBV genome in the PCR mixture (or 1.3×103 to 6.7× 103 copies/ml serum). Fewer than 50 copies were not detected reliably by these assays due to the formation of primer artefacts. Quantification was found to be inaccurate for the 50 copies standard as judged by the loss of linearity of the log input copy number

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Table 2 Reproducibility of the HBV quantitative real-time PCR Strain

Mean (log10)

Range (log10)

Standard deviationa

Standard deviation for extractsb

Standard deviation for PCR runsc

95-23426 95-23474 95-3475 95-23476 95-23480

10.01 8.14 7.99 9.30 7.61

9.84–10.29 8.00–8.26 7.84–8.14 9.15–9.43 7.26–7.86

0.18 0.10 0.12 0.12 0.22

0.06 0.05 0.02 0.02 0.09

0.18 0.05 0.07 0.12 0.23

a

Standard deviation for all the measurements. Standard deviation for the average measurement of each extract. c Standard deviation for the average measurement of each PCR run. b

versus cycle number at threshold crossing plot. For this reason, the lower limit for quantification was set at 102 copies (1.3×104 copies/ml serum) for the single round PCR assay. The nested assay was designed to overcome this limitation in sensitivity and was applied to all samples found to contain B1.3× 104 HBV genomes/ml serum. The nested assay is sensitive to the level of a single HBV genome in the PCR reaction giving a detection limit of :25 HBV genomes/ml. Quantification was found to be reliable down to a level of : 4× 102 genomes/ml.

3.2. Inhibitory samples

normally distributed on the logarithmic scale. Table 2 shows the average values and standard deviations (log10 scale) for the five samples. The reproducibility of the quantitative PCR was further estimated by measurement of the levels of HBV genomes in two separate extracts of sera from 37 patients (Fig. 2). The range of values of genome equivalents per ml was from 103 to 2× 109 and the average log10 difference between the two values was 0.45. Multiple extracts of a standard serum (Centraal Laboratorium van de Bloedtransfusiedienst) nominally containing 3×104 copies of HBV DNA/ml were made. These samples were tested in either the unnested (24 samples) or the nested PCR (27

Samples containing inhibitors of the PCR process could be detected by the absence of product accumulation in the F1 (540 nm) detection channel. Samples were also considered inhibitory to the PCR if the slope describing product accumulation (channel F1, 540 nm) during the linear phase was shallow relative to the adjacent standards i.e. if the maximum gradient of the curve describing product accumulation was less than half of that of the nearest standard.

3.3. Reproducibility of HBV 6iral load estimation by real-time PCR The reproducibility of the surface gene quantitative PCR was assessed using three separate viral DNA extracts from each of five serum samples. Each extract was then tested in duplicate in three different PCR runs. The values were found to be

Fig. 2. Duplicate analyses of 37 sera in the quantitative real-time PCR. Duplicate aliquots of 37 patient sera were tested. The range was from 103 to 2 ×109 HBV genomes/ml and the mean log10 difference between duplicates was calculated to be 0.45.

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Table 3 The accuracy of the HBV quantitative PCR for VQCa specimens VQC sample

VQC expected

Real-time PCR observed

2 3 5 7 8 9 10 11 12 13 14 15 16 17 18 19 21 22 23 24

37 300 0 113 437 0.4 3730 43 700 37 11 300 4 373 132 44 0 1130 0.4 0 11 0 1320

41 400 B400 B400 B400 B400 2150 43 400 B400 4630 B400 B400 B400 B400 B400 886 B400 B400 B400 B400 419

a Viral quality control (VQC) specimens were from Centraal Laboratorium van de Bloedtransfusiedienst, Amsterdam.

samples). The average values determined for the extracts were adjusted to 3× 104 copies/ml for the unnested and nested reactions by recalibration of the plasmid standards. Recalibrated standards were used in all subsequent quantitative PCRs. To calculate standard deviations the values were converted to the log10 scale giving values of 0.588 and 0.268 for nested and unnested PCRs, respectively.

below the test cut-off. The remaining sample, which nominally contained 437 HBV genomes/ml also, gave a value below the cut-off of the quantitative PCR. No significant difference in copy number estimates was found when the preparation of plasmid standard used in the experiments described above were used in either native or linearised forms. This was probably because these preparations contained significant quantities of open circular and linear plasmid DNA.

3.5. Comparison between real-time PCR and the Roche Monitor kit Quantitative HBV load values were obtained for 39 surface antigen positive blood donor sera using both the real-time PCR and the Roche Monitor kit (Fig. 3). The mean difference between the two assays for samples that gave values above the cut-off in both tests (36 samples) was 0.57 log10 (range, 0.018 –1.53 log10). The real-time PCR gave a higher value for 21 of these 36 samples. The mean difference for samples that gave values of \ 1× 106 (11 samples) was 0.64 log10 (range, 0.14 –0.96 log10). For each of these samples, the real-time PCR reported a higher value than the Monitor kit. A positive sample estimated to contain 1.8× 107 viral copies/ml was diluted in HBV negative human serum to 3.7× 106, 3.7× 105 and 3.7×104 copies/ml. The four samples were tested in the real-time PCR and in the Roche Monitor kit (Fig. 4). The Monitor kit gave lower than expected signals at high virus concentrations.

3.4. Accuracy of HBV 6iral load estimation by real-time PCR 4. Discussion The accuracy of the quantitative PCR was assessed by analysis of the levels in samples from the Viral quality control (VQC) HBV-DNA panel (Centraal Laboratorium van de Bloedtransfusiedienst). Six samples gave values above the cut-off threshold of the test (Table 3). All the values determined by quantitative PCR were within 0.5 log10 of the nominal values with a mean difference of 0.18 log10. Thirteen samples containing B400 genome equivalents per ml gave values

4.1. Real-time PCR for quantifying HBV The rate of product accumulation during PCR cycling passes through several phases. Initially, amplification is close to exponential with the template doubling with each completed cycle. Later, several factors combine to reduce the efficiency of amplification including the depletion of resources (Taq polymerase, dNTPs, primers) within the

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mixture and the tendency of the product to renature during annealing rather than hybridising to the primers. In the final stages of PCR the accumulation of product may cease and then be reversed as newly synthesised copies fail to replace those lost due to hydrolysis. Quantification by real-time PCR relies on a detection system that is able to measure the amount of product at a convenient point in each temperature cycle while the process is still in the exponential phase and with sufficient sensitivity for the product to be detected. The initial copy number can then be calculated from the cycle number at which a detection threshold is crossed. This calculation should take place while the PCR amplification is virtually exponential because in the latter stages of PCR cycling the quantity of product is not proportional to the initial template copy number. For the Bi-probe system it is important that the PCR product is relatively short (: 100 bp) in order to minimise the emission of photons from SYBR-green I molecules bound to reannealed PCR product. A proportion of photons emitted by SYBR-green I will be at the trailing (low

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energy) edge of the emission spectrum and will be measured in the LightCycler™ channel F2, which detects long wavelength light. The intensity of signal generated from this source is dependent upon the quantity of double stranded DNA produced and is not, therefore, sequence specific. For the Bi-probe PCRs reported here the majority of fluorescence in the F2 channel during product accumulation resulted from fluorescent resonance energy transfer between SYBR-green I and Cy5. The signal threshold was set at a level that was not crossed by PCR reactions that were negative i.e. the low level increase in F2 fluorescence contributed by the binding of SYBR-green I independent of the probe was insufficient to allow threshold crossing. The assay is based on a standard curve derived from samples containing known numbers of plasmid copies carrying the sequence amplified in the PCR. Since a standard curve must be produced for every run the number of points used (five) is a compromise between accuracy and practicability. This LightCycler™ based test is similar to one recently described by Abe et al. (1999) employing Taq Man chemistry (Heid et al., 1996).

Fig. 3. Comparison of real-time PCR and the Monitor kit. Real-time PCR and the Roche Monitor kit were used to obtain quantitative HBV virus concentration values for 39 HB surface antigen positive blood donor sera.

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Fig. 4. Comparison of the real-time PCR and the Monitor kit for a dilution series. A single HBV positive serum specimen was diluted with HBV negative serum to 1.8 × 107, 3.7 × 106, 3.7 ×105 and 3.7 ×104 copies/ml was tested in both quantitative assays. () the expected values, ( … … ) Roche Monitor kit estimates, and ("…– …"…–…") real-time PCR estimates.

4.2. Sensiti6ity and range of the assay The single round surface gene quantitative PCR was able to detect approximately 50 copies of target sequence cloned into the pTOPO plasmid. The nested assay was able to detect a single copy of the cloned surface gene introduced into the reaction mixture. The lower limits for accurate quantification were higher and were set at 100 and 15 copies for the single round and nested PCRs, respectively, i.e. 13 000 and 400 genomes/ml serum. For the nested assay the quantitative accuracy in the lower range of viral loads was limited by sampling variation. In contrast, the main limitation on the lower range accuracy of the single round PCR was variation among reactions since these resulted in differences in the accumulation kinetics of primer artefacts. Reactions that favour primer artefact formation will appear to contain lower target copy numbers than reactions in which this process is inhibited. The upper limit for the dynamic range of the assay was effectively determined by the range of standards chosen. The upper value of the range was set 1 log10 higher than the highest standard i.e. equivalent to 1.3× 1010 and 1.3×108 copies/ ml serum for the standard and nested reactions, respectively. The quantitative range could be extended by several log10 units if suitable standards

were run. However, a baseline fluorescence must be established prior to detectable accumulation of product. If this is not possible the sample is beyond the upper limit for quantification in this system without prior dilution.

4.3. Reproducibility and accuracy of the real-time PCR The reproducibility of DNA extraction was shown to be high in comparison with the reproducibility of the quantitative PCR since the standard deviations for analysis of different extracts were higher than the standard deviations for PCR runs (Table 2). Improvements in the accuracy of the quantitative PCR for a single sample can therefore be expected to be achieved most effectively by performing multiple analyses on a single extract. Nevertheless, in clinical settings where serial patient specimens are available a single measurement on each sample may provide an adequate indication of the trend in viral concentration. The reproducibility of quantification for samples containing viral concentrations between 103 and 2 × 109 genomes/ml was good (Fig. 2). However, on average, larger log10 differences between duplicate measurements were found for samples in the lower part of the range. This is to be expected since more amplification cycles are

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required before the threshold is crossed for these samples. Differences in amplification efficiency will therefore affect the weaker samples more. Furthermore, the effect of sample variation will be greater for low concentration specimens. Repeat analysis of the VQC specimen using both the standard and the nested versions of the assay showed the lower reproducibility of the nested PCR. This is attributable to the additional manipulations required. The accuracy of viral load estimation was tested using a range of VQC standards containing between zero and 4.37×104 viral genomes. The differences between the nominal and measured values (Table 3) were of the same order as those between duplicate measurements of virus concentration in a single sample. This result indicates that the calibration of the system was satisfactory.

4.4. Comparison between the results of HBV real-time PCR and the Roche HBV Monitor kit Between the two methods there was good general correspondence for results in the range, 400 – 105 genomes /ml serum. However, at higher viral loads (\ 106/ml) the kit consistently under-estimated the concentration (Fig. 3). This result was confirmed by analysis of serum dilutions (Fig. 4). These results are consistent with the findings of a study by Pawlotsky et al. (2000) who compared the Monitor kit with two hybridisation methods.

4.5. Costs Extraction of HBV DNA from sera and realtime quantitative PCR can be completed in approximately 2 h for the single round PCR version of the test or in approximately 4 h if it is necessary to perform the nested PCR. Reagent costs are relatively low for the single round PCR due to the small reaction mixture volumes required in the LightCyclerTM. Both versions of the test, unnested and nested, are considerably less expensive than the current version of the Monitor kit. A major advantage of real-time PCR for quantification of HBV is its accuracy and reliability over a wide range of concentrations. Few samples have concentrations above the upper limit of the

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assay (1.3× 1010 copies/ml), and for the rest, the LightCycler™ assay may be performed rapidly and conveniently. References Abe, A., Inoue, K., Tanaka, T., Kato, J., Kajiyama, N., Kawaguchi, R., Tanaka, S., Yoshiba, M., Kohara, M., 1999. Quantitation of hepatitis B virus genomic DNA by real-time detection PCR. J. Clin. Microbiol. 37, 2899– 2903. Beasley, R.P., 1988. Hepatitis B virus – the major etiology of hepatocellular carcinoma. Cancer 61, 1942– 1956. Boom, R., Sol, C.J.A., Heijtink, R., Wertheim-van Dillen, P.M.E., van der Norordaa, J., 1991. Rapid purification of hepatitis B virus DNA from serum. J. Clin. Microbiol. 29, 1804– 1811. Dienstag, J.L., Perillo, R.P., Schiff, E.R., Batholomew, M., Vicary, C., Rubin, M., 1995. A preliminary trial of lamivudine for chronic hepatitis B infection. N. Engl. J. Med. 333, 1657– 1661. Heid, C.A., Stevens, J., Livak, K.J., Williams, P.M., 1996. Real-time quantitative PCR. Genome Res. 6, 986– 994. Hendricks, D.A., Stowe, B.J., Hoo, B.S., Kolberg, J., Irvine, B.D., Neuwald, P.D., Urdea, M.S., Perrillo, R.P., 1995. Quantitation of HBV DNA in human serum using branched DNA signal amplification assay. Am. J. Clin. Pathol. 104, 537– 546. Kessler, H.H., Pierer, K., Dragon, E., Lackner, H., Santner, D., Stunzner, D., Stelzl, E., Waitz, B., Marth, E., 1998. Evaluation of a new assay for HBV DNA quantitation in patients with chronic hepatitis B. Clin. Diagn. Virol. 9, 37 – 43. Lai, C.L., Chien, R.N., Leung, N.W., Chang, T.T., Guan, R., Tai, D.L., Ng, K.Y., Wu, P.C., Dent, J.C., Barber, J., Stephenson, S.L., Gray, D.F., 1998. A one-year trial of lamivudine for chronic hepatitis B. Asia hepatitis Lamivudine Study Group. N. Engl. J. Med. 339, 61 – 68. Pawlotsky, J.-M., Bastie, A., He´zode, C., Lonjon, I., Darthuy, D., Re´mire´, J., Dhumeaux, D., 2000. Routine detectin and quantification of hepatitis B virus DNA in clinical laboratories: performance of three commercial assays. J. Virol. Meth. 85, 11 – 21. Perrillo, R.P., 1991. Treatment of chronic hepatitis B. In: Hollinger, F.B., Lemon, S.M., Margolis, H.S. (Eds.), Viral hepatitis and liver disease. Williams & Wilkins, Baltimore, pp. 616– 623. Noborg, U., Gusdal, A., Pisa, E.K., Hedrum, A., Lindh, M., 1999. Automated quantitative analysis of hepatitis B virus DNA by using the Cobas Amplicor HBV Monitor test. J. Clin. Microbiol. 37, 2793– 2797. Wong, D.H.K., Cheung, A.M., Orourke, K., Naylor, C.D., Detsky, A.S., Heathcote, J., 1993. Effect of alpha-interferon treatment in patients with hepatitis B e antigen-positive chronic hepatitis B: a meta-analysis. Ann. Int. Med. 119, 312– 323.