Fully Automated, Internally Controlled Quantification of Hepatitis B ...

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2004, p. 585–590 0095-1137/04/$08.00⫹0 DOI: 10.1128/JCM.42.2.585–590.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Vol. 42, No. 2

Fully Automated, Internally Controlled Quantification of Hepatitis B Virus DNA by Real-Time PCR by Use of the MagNA Pure LC and LightCycler Instruments Victoria Leb,1 Markus Sto ¨cher,1 Elizabeth Valentine-Thon,2 Gabriele Ho ¨lzl,1 Harald Kessler,3 1 1 Herbert Stekel, and Jo ¨rg Berg * Institute of Laboratory Medicine, General Hospital Linz, A-4020 Linz,1 and Molecular Diagnostics Laboratory, Institute of Hygiene, Karl-Franzens-University Graz, A-8010 Graz,3 Austria, and Laboratory Dr. M. Sandkamp, B. Ko ¨ster, Dr. R. Hiller, 28259 Bremen, Germany2 Received 28 April 2003/Returned for modification 14 July 2003/Accepted 22 October 2003

We report on the development of a fully automated real-time PCR assay for the quantitative detection of hepatitis B virus (HBV) DNA in plasma with EDTA (EDTA plasma). The MagNA Pure LC instrument was used for automated DNA purification and automated preparation of PCR mixtures. Real-time PCR was performed on the LightCycler instrument. An internal amplification control was devised as a PCR competitor and was introduced into the assay at the stage of DNA purification to permit monitoring for sample adequacy. The detection limit of the assay was found to be 200 HBV DNA copies/ml, with a linear dynamic range of 8 orders of magnitude. When samples from the European Union Quality Control Concerted Action HBV Proficiency Panel 1999 were examined, the results were found to be in acceptable agreement with the HBV DNA concentrations of the panel members. In a clinical laboratory evaluation of 123 EDTA plasma samples, a significant correlation was found with the results obtained by the Roche HBV Monitor test on the Cobas Amplicor analyzer within the dynamic range of that system. In conclusion, the newly developed assay has a markedly reduced hands-on time, permits monitoring for sample adequacy, and is suitable for the quantitative detection of HBV DNA in plasma in a routine clinical laboratory. hibit greater accuracy, provide extended ranges of linearity compared to those of conventional PCR assays, and allow PCR product detection and quantification in a closed system (1, 7, 23, 29). In the LightCycler instrument, for example, PCR is performed in glass capillaries that are heated and cooled by a computer-controlled fan and heating coil, thereby markedly accelerating amplification (35, 36). For PCR product detection, various formats of fluorescence dye technologies can be used, such as the TaqMan technology or paired hybridization probes that are designed to exhibit fluorescence resonance energy transfer (FRET) upon hybridization (35). LightCycler protocols have been developed for the detection and quantification of HBV DNA in serum and plasma (6, 16, 28). While these assays are rapid and sensitive and show a linearity range of several orders of magnitude, they do not provide measures for monitoring for sample adequacy and are not automated beyond the degree offered by the LightCycler instrument itself. Clinical specimens may contain inhibitors of PCR amplification (2). As such inhibitors are not always reliably removed during DNA purification, ICs have been introduced into PCR assays. Those ICs that are coamplified with the virus-specific nucleic acids by use of the same set of primers and within the same reaction vessel are thought to exhibit the most accurate control of the amplification reaction and are suitable for the identification of inhibitors of the PCR (4, 30, 31). The demand for the molecular-level testing of specimens from patients with chronic viral infections has increased in recent years (3). To meet this demand and to avoid human error in the assay workup, attempts are being made to completely automate molecular assays for routine clinical labora-

The use of molecular assays for the detection and quantification of hepatitis B virus (HBV) DNA in serum or plasma has become a standard laboratory approach for the diagnosis of HBV infection and the management of disease in HBV-infected patients. Measurements of HBV DNA levels are routinely used to identify infectious chronic carriers and to predict and monitor the efficacies of antiviral treatment regimens (5, 15, 19, 25). In addition, when serological testing may be inconclusive for the diagnosis of an HBV infection, e.g., due to the presence of genetic variants of HBV, the detection of HBV DNA has been shown to be useful in resolving those uncertainties (34). Several assays for the detection and quantification of HBV DNA have been described. They are based on various molecular principles such as branched DNA technology, direct hybridization and signal amplification, or PCR (13, 14, 17, 21, 22). One of these, the Cobas Amplicor HBV Monitor test, is a PCR-based, commercially available, standardized assay now widely used in routine laboratories. The assay allows automated PCR amplification, product detection, and quantification (8, 18, 26, 27). The assay includes an internal amplification control (IC) for monitoring for sample adequacy as well as for quantitative assessment (8). The quantification of HBV DNA in clinical specimens has significantly improved with the introduction of real-time PCR into routine diagnostic laboratories. Real-time PCR assays ex* Corresponding author. Mailing address: Institute of Laboratory Medicine, General Hospital Linz, Krankenhausstrasse 9, A-4020 Linz, Austria. Phone: 0043-70-7806-1174. Fax: 0043-70-7806-1815. E-mail: [email protected]. 585

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tories. This includes the nucleic acid purification step, as this is the most labor-intensive part of molecular assays. Several instruments for automated nucleic acid purification have been brought to the market (10, 12 ), including the MagNA Pure LC instrument (Roche Applied Science, Mannheim, Germany). This instrument has recently been evaluated (10, 11, 20, 24). In this study we describe the development of a fully automated real-time PCR (HBV LC-PCR) assay for the quantitative detection of HBV DNA in plasma with EDTA (EDTA plasma). The MagNA Pure LC instrument is used for automated DNA purification and PCR mixture preparation. Realtime PCR is performed on the LightCycler instrument. To monitor the reaction for inhibitors of the PCR, an IC was constructed as a PCR competitor and was introduced into the assay at the stage of DNA purification. The newly developed HBV LC-PCR assay was evaluated by analyzing eight samples from the European Union Quality Control Concerted Action (EU QCCA) HBV Proficiency Panel 1999 (32) and by assaying 123 EDTA plasma samples that had previously been assessed by the Cobas Amplicor HBV Monitor test in an accredited routine diagnostic laboratory. MATERIALS AND METHODS Materials and specimens. An HBV reference standard (standard 1872/01; 50,000 IU/ml) was generously provided by M. Nu ¨bling, Paul Ehrlich Institute, Langen, Germany. This reference standard was calibrated against the World Health Organization standard at the Paul Ehrlich Institute by replicate analysis by both the Cobas Amplicor HBV Monitor test and an in-house qualitative HBV-PCR (M. Chudy, unpublished data). Limiting dilutions of the materials were tested by the in-house HBV-PCR, and the results of replicate tests were statistically evaluated by probit analysis. Both test methods revealed similar potencies and allowed expression of the amount of the reference standard in international units per milliliter. The HBV reference standard was spiked in graded amounts into HBV surface antigen-negative EDTA plasma to determine the detection limit of the assay. HBV DNA-positive EDTA plasma was calibrated against the HBV reference standard and was used as an external quantification standard in the LightCycler assay. An IC was generated and added to all samples prior to DNA purification. For evaluation of the novel assay, the eight samples of the EU QCCA HBV Proficiency Panel 1999 and 123 plasma samples that were quantitatively assayed for HBV DNA by the Cobas Amplicor HBV Monitor test were used. EDTA plasma samples from patients with no clinical symptoms or laboratory findings of infectious diseases were tested as negative control samples. HBV DNA-specific IC. An HBV-specific IC was devised as a PCR competitor and consisted of the HBV-specific forward and reverse primer sequences flanking a stretch of heterologous DNA. The IC was constructed as described recently (30, 31). Briefly, a stretch of the bacterial neomycin phosphotransferase (neo) gene (GenBank accession number V00618; bp 473 to 603) was amplified by conventional preparative PCR by using composite primers that consisted of the HBV- and the neo-specific sequences 5⬘-GACCACCAAATGCCCCTATCT CCTGCCGAGAAAGTATCCA-3⬘ (GenBank accession numbers NC003977 [bp 2298 to 2316 are underlined] and V00618 [bp 473 to 493], respectively) and 5⬘-CGAGATTGAGATCTTCTGCGACGCACCGGCTTCCATCCGA-3⬘ (GenBank accession numbers NC003977 [bp 2415 to 2436 are underlined] and V00618 [bp 588 to 603], respectively). The construct obtained (173 bp) was cloned into the PCR II-TOPO plasmid vector by using the TOPO TA cloning kit (Invitrogen Ltd, Paisley, United Kingdom) according to the instructions of the manufacturer. The plasmid DNA was purified from the transformed bacteria by a standard miniprep procedure and linearized by HindIII restriction. The DNA concentration was assessed by UV spectrophotometry. The IC fragment was subjected to automated sequence analysis, which confirmed the expected DNA sequence. DNA preparation. DNA from EDTA plasma samples (200 ␮l each) was automatically purified on the MagNa Pure LC instrument (software version 2.1) with the MagNa Pure LC Total Nucleic Acid Isolation kit (Roche Diagnostics GmbH) according to the instructions of the manufacturer. The purification runs were performed with the MagNA Pure LC program Total-NA-HBV-08 (Roche

J. CLIN. MICROBIOL. Applied Science, Penzberg, Germany). Prior to DNA purification, 20 ␮l of proteinase K solution (20 mg/ml) was added, and the mixture was incubated for 3 min at room temperature. Thereafter, IC DNA was spiked into the samples to yield 5,000 copies/ml, and this was immediately followed by automated DNA purification. Elution was performed with 50 ␮l of elution buffer. All purification runs included one previously tested negative control plasma sample, which was also complemented with IC DNA. HBV DNA-specific LightCycler PCR. The HBV DNA-specific LightCycler PCR was established as a competitive PCR to amplify both HBV DNA and IC DNA with the HBV-specific primers in the same capillary. For the HBV DNA amplification, the C gene of HBV was targeted by using primers with the sequences 5⬘-GACCACCAAATGCCCCTAT-3⬘ and 5⬘-CGAGATTGAGATCTT CTGCGAC-3⬘ (GenBank accession number NC003977; bp 2298 to 2316 and 2415 to 2436, respectively), which have been described previously (16). The HBV DNA PCR products were detected with FRET hybridization probes with the sequences 5⬘-GA(G/C)GCAGGTCCC-CTAGAAGAAGAA-3⬘-FL (where FL represents fluorescein) and LC-Red-640-5⬘-TCCCTCGCCTCGCAGACG (A/C)AG(A/G)TCTC-p–3⬘ (where p represents phosphorylation) (GenBank accession number NC003977; bp 2355 to 2378 and 2380 to 2410, respectively). The IC DNA PCR products were detected with FRET hybridization probes with the sequences 5⬘-GCTGCATACGCTTGATCCGGCT-3⬘-FL and LC-Red-705-5⬘-C CTGCCCATTCG-ACCACCAAGC-p–3⬘ (GenBank accession number V00618; bp 516 to 537 and 539 to 560, respectively). The primers were obtained from MWG Biotech, Ebersberg, Germany. The hybridization probes were obtained from TIB MOLBIOL, Berlin, Germany. The PCR mixture was prepared with LightCycler Fast Start DNA Master Hybridization Probes (Roche Diagnostics GmbH). A primer-probe mixture (13 ␮l/PCR mixture) was prepared. The mixture contained MgCl (final concentration, 4 mM), HBV DNA-specific primers (0.4 ␮M each), and the HBV DNA- and IC DNA-specific FRET hybridization probes (0.2 ␮M each). A buffer-enzyme mixture (2 ␮l/PCR mixture) was prepared to the concentrations described in the manufacturer’s instructions. Prior to DNA purification, the primer-probe mixture, the buffer-enzyme mixture, the quantification standards, and the LightCycler carousel containing the capillaries used in the PCR were placed into the cooled postelution section of the MagNA Pure LC instrument. After DNA purification, the postelution program of the MagNA Pure LC instrument was used to automatically combine the primer-probe mixture with the buffer-enzyme mixture. The resulting reaction mixture (15 ␮l) and the sample DNAs (5 ␮l) were then automatically distributed to the LightCycler capillaries. The capillaries were manually closed, and the carousel was spun (15 s at 3,000 rpm) and placed into the LightCycler instrument. PCR was performed with the following cycling program: 95°C for 9 min, followed by 50 cycles of 2 s at 95°C, 10 s at 60°C, and 15 s at 72°C. Thereafter, the products were analyzed by applying 95°C for 1 min, 57°C for 2 min, and 50°C for 2 s, followed by an increase in the temperature from 50 to 85°C (at 0.2°C/s), with continuous recording of the fluorescence. Data acquisition was performed by applying the following fluorescence channel settings: F2 for the HBV DNA products and F3 for the IC DNA products (9, 35). The melting temperatures for the HBV DNA- and IC DNAspecific FRET hybridization probes were both 68°C. A color compensation file was generated according to the manufacturer’s instructions and was used in all PCR runs. All PCR runs were accompanied by three quantification standards, a negative control consisting of noninfectious EDTA plasma from a healthy individual containing the same amount of IC DNA used in the clinical samples, and a reagent control that comprised PCR-grade water instead of sample DNA. Throughout the study version 3.5.3 of the LightCycler software was used. Cobas Amplicor HBV Monitor test. Version 2.0 of the Cobas Amplicor HBV Monitor test (Roche Applied Science, Mannheim, Germany) was used. The assay was performed according to the manufacturer’s instructions in the Department of Molecular Biology (former head, E. Valentine-Thon) in the former Laboratory Dr. Schiwara and Partners (now Medical Laboratory Bremen), Bremen, Germany. The test sensitivity is claimed to be 200 HBV copies/ml, and the linear dynamic range is reported to be 2 ⫻ 102 to 2 ⫻ 105 HBV copies/ml. For all values ⱖ2 ⫻ 105 HBV copies/ml, Roche recommends dilution and retesting of the sample. The software reports the results obtained by this assay as ⬍200 copies/ml when no copy number is detected, ⬍200 copies/ml when HBV copy numbers of ⬍200/ml are detected, or as the actual copy numbers in the samples when the amounts exceed 200 copies/ml. In samples with ⱖ108 HBV copies/ml, the IC provided with the kit fails to be amplified and the result is declared “invalid” by the software (33). For this study, such samples were either diluted 1:100 and retested to obtain a valid copy number or reported as ⱖ108 HBV copies/ml.

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TABLE 2. Results obtained with the EU QCCA HBV Proficiency Panel 1999

TABLE 1. Determination of detection limit No. of HBV DNA copies/ml

No. of tests with positive results/no. of tests

%

1,000 800 400 200 100 50

9/9 9/9 9/9 9/9 7/9 5/9

100 100 100 100 78 56

Data analysis. The Spearman rank test was used to calculate correlation coefficients for assumed correlations between the quantification results obtained by the Cobas Amplicor HBV Monitor test and the HBV LC-PCR assay.

RESULTS The detection limit of the HBV LC-PCR assay was determined as the first step in the study. The HBV reference standard was spiked in graded amounts into HBV-negative plasma samples containing 5,000 IC DNA copies/ml. After automated DNA isolation and HBV LC-PCR, the detection limit of the HBV LC-PCR assay was found to be 200 HBV copies/ml, with 100% reproducibility (Table 1). Inconsistent positive and negative results were obtained when samples with ⬍200 HBV copies/ml were assayed. The IC was amplified in all samples used for determination of the detection limit. In the second step, the dynamic range of the HBV LC-PCR assay was examined by using a log-fold dilution series of a positive HBV plasma sample containing 8 ⫻ 108 HBV DNA copies/ml. Each sample of the dilution series also contained IC DNA (5,000 copies/ml). After automated DNA purification, HBV LC-PCR was performed. As shown in Fig. 1, the HBV LC-PCR assay exhibited a linearity of 8 orders of magnitude. In the third step, the precision of the HBV LC-PCR assay was determined by assessing the intra- and interassay coefficients of variation (CV). For the former, the crossing-point values of the HBV- and IC-specific amplifications from 10 replicates of an HBV DNA-positive plasma sample (with approximately 25,000 copies of HBV DNA/ml and 5,000 copies of IC DNA/ml) were used. The intra-assay CVs were 0.7% for

Panel member no.

No. of target HBV DNA copies/ml

1 2 3 4 5 6 7 8

10,000,000 Negative 2,000,000 1,000 2,000,000 200,000 Negative 10,000,000

a

No. of HBV DNA copies/mla Test 1

Test 2

10,360,000 ⬍200 420,000 410 1,506,000 80,730 ⬍200 4,548,000

8,961,000 ⬍200 317,900 856 1,284,000 87,100 ⬍200 2,345,000

Results obtained by the HBV-LC-PCR assay.

the HBV amplification and 1.45% for the IC DNA amplification (data not shown). For the interassay CVs, the crossingpoint values for amplification of HBV and IC DNA from the aforementioned HBV DNA-positive plasma samples of 10 different PCR runs were used. The interassay CVs were 1.48% for HBV DNA amplification and 2.26% for IC DNA amplification (data not shown). The eight members of the EU QCCA HBV Proficiency Panel 1999 were examined as a first validation of the HBV LC-PCR assay. The quantities obtained were compared with the quantities in the proficiency panel (Table 2). The results were in acceptable agreement with the HBV DNA concentrations reported for the samples in the panel. The two panel members not containing HBV DNA were also negative by the HBV LC-PCR assay. For a second evaluation of the HBV LC-PCR assay, 123 clinical samples were used. The test results were compared with the results obtained by the Cobas Amplicor Monitor HBV test as the reference method (Table 3; Fig. 2 and 3). Of the 123 samples, 113 yielded a positive result by the Cobas Amplicor Monitor HBV test, and 100 of these 113 samples were found to contain ⬎200 HBV DNA copies/ml. These 100 positive samples were also found to contain ⬎200 HBV DNA copies/ml by the HBV LC-PCR assay (Table 3). Of the 13 samples found to contain ⬍200 HBV DNA copies/ml by the Cobas Amplicor Monitor HBV test, 5 samples also tested positive by the HBV LC-PCR assay. Of these five samples, four contained ⬎200 HBV DNA copies/ml and one contained ⬍200 copies/ml (Table 3). The Cobas Amplicor Monitor HBV test did not detect any HBV DNA in 10 samples. The same result was also found by the HBV LC-PCR assay for 7 of these 10 samples, whereas

TABLE 3. Results for 123 EDTA plasma samples tested by the HBV LC-PCR assay and the Cobas Amplicor HBV Monitor test

Result for samples tested by HBV LC-PCR assay

FIG. 1. Linearity of the HBV LC-PCR assay. Quantitation results for a log-fold serial dilution of an HBV DNA-positive plasma sample were plotted against the PCR cycle number. Logarithmic linear regression was performed (y ⫽ ⫺3.7x ⫹ 47.3; r ⫽ 0.99).

HBV DNA not detected HBV DNA detected at ⬍200 copies/ml HBV DNA detected at ⬎200 copies/ml

No. of samples with the following result by Cobas Amplicor HBV Monitor test: HBV DNA not detected

HBV DNA detected at ⬍200 copies/ml

HBV DNA detected at ⬎200 copies/ml

7 0

8 1

0 0

3

4

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J. CLIN. MICROBIOL. FIG. 2. Fluorescence versus cycle number plots for clinical samples showing HBV-specific amplifications (A) and the corresponding IC-specific amplifications (B). Standards 1, 2, and 3 contained 50,000, 5,000, and 500 HBV DNA copies/ml, respectively; and each standard contained IC DNA. Patients 1 and 2 represent the sources of two different EDTA plasma samples. Note that the IC DNA was not amplified from the sample from patient 1 due to competition with the large amounts of HBV DNA. Noninfectious EDTA plasma was used as a negative control, complemented with IC, and copurified with patient samples on the MagNA Pure LC instrument. PCR-grade water was used as a reagent control and was directly introduced into the HBV LC-PCR.

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FIG. 3. Correlation between the amounts of HBV DNA measured in plasma samples by the HBV LC-PCR assay and the Cobas Amplicor HBV Monitor test (the regression equation was y ⫽ 0.814x ⫹ 0.994; Spearman rank test, r ⫽ 0.946 and P ⬍ 0.0001; 95% confidence interval, 0.919 to 0.965). Only those 97 plasma samples that returned a quantitation result between 108 and 200 HBV DNA copies/ml by the Cobas Amplicor HBV Monitor test were used for the correlation.

3 samples tested positive by the HBV LC-PCR assay, with all 3 samples exhibiting ⬎200 HBV DNA copies/ml (Table 3). When the quantitation results obtained by the Cobas Amplicor Monitor HBV test for 97 of the 100 samples with ⬎200 HBV DNA copies/ml were compared with the quantitation results obtained by the HBV LC-PCR assay, similar HBV DNA copy numbers were obtained (Fig. 3). The quantitation results obtained by both assays were in good overall agreement and were found to be significantly correlated with each other (Spearman rank test, r ⫽ 0.946 and P ⬍ 0.0001; 95% confidence interval, 0.919 to 0.965). Three of the 100 samples found to contain ⬎200 HBV DNA copies/ml by the Cobas Amplicor Monitor HBV test were reported to contain ⬎108 HBV copies/ ml, and therefore, the results were not compared with those obtained by the HBV LC-PCR assay. In the assessment of the 123 clinical samples by the HBV LC-PCR assay, the IC DNA was always amplified to detectable levels and to the expected levels when the sample contained ⬍50,000 HBV DNA copies/ml. This result was obtained for 76 of the 123 clinical samples (Fig. 3). When the quantification results exceeded approximately 50,000 HBV DNA copies/ml, the amplification of the IC DNA was competitively inhibited. This occurred with 47 of the 123 clinical samples (data not shown). When 16 samples including 1 negative control sample were subjected to DNA purification on the MagNA Pure LC instrument and the HBV LC-PCR assay was subsequently carried out with three quantification standards, the hands-on time required was only approximately 30 min, and the entire HBV LC-PCR assay was completed within 3 h. DISCUSSION In this report we describe a newly developed fully automated assay for the quantitative detection of HBV DNA in EDTA

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plasma with the MagNA Pure LC instrument for the automated purification of HBV DNA and the automated preparation of PCR mixtures and with the LightCycler instrument for real-time PCR amplification and quantification. The new assay uses an IC to monitor sample adequacy. We have demonstrated that this assay exhibits a high degree of precision and a wide range of linearity, allowing detection of HBV DNA at levels ranging from 200 to approximately 109 copies/ml without the need for sample dilution or concentration. The precision and the linearity of the assay were comparable to those previously reported for real-time PCR assays based on both the TaqMan and the LightCycler technologies (1, 6, 16, 28, 29). In clinical diagnostic PCR assays, the use of ICs is necessary to demonstrate the absence of PCR inhibition (2). As PCR inhibitors are not always reliably removed from clinical specimens during DNA purification, they can diminish the quantification results and/or induce false-negative test results. For this reason, the HBV LC-PCR assay was provided with an IC that was added to the samples at the stage of DNA purification. The IC DNA was then amplified together with the HBV DNA by the set of HBV DNA-specific primers in the same capillary. Thus, the assay was controlled from the DNA purification step through the amplification and product detection steps. The IC DNA products were detected with IC DNAspecific FRET hybridization probes labeled with a fluorochrome different from the ones used for the detection of HBV DNA products. By using the two separate fluorescence channels (F2 and F3) of the LightCycler instrument devised for the detection of FRET hybridization probes, the detection of the IC DNA-derived products could be performed independently from the detection of the HBV DNA-derived products (9). Cross talk between the two fluorescence channels was not observed during this study. The IC DNA was amplified in a competitive fashion together with potential HBV DNA. This did not adversely influence the linearity of the assay, presumably due to the lower amplification efficiency of the IC compared to the amplification efficiency of the HBV DNA as well as to the increased amounts of primers used in the competitive HBV LC-PCR compared to the amounts used in an HBV LC-PCR without an IC. The competitive nature of the HBV LC-PCR assay allowed detectable IC DNA amplification, however, only when a sample contained less than approximately 50,000 HBV DNA copies/ml. The IC was competitively inhibited when higher HBV DNA concentrations were present. Similar competitive inhibition has been reported for the IC in the Cobas Amplicor HBV Monitor test (34). Thus, while potential PCR inhibitors cannot be identified in highly viremic samples by this assay, PCR inhibition in all samples with low levels of viremia or truly negative samples can be adequately monitored. Since manual purification of nucleic acids from clinical samples represents the most labor-intensive, contamination-prone part of a molecular assay, attempts have been made to automate these steps. Use of the MagNA Pure LC instrument in conjunction with the HBV LC-PCR assay proved to be a markedly labor-saving means of testing, as others have described previously (10, 11, 20). Cross-contamination was not observed during this study. Furthermore, the preparation and distribution of both the PCR mixture and the isolated sample DNA were conveniently carried out in an automated fashion. The

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high precision of the HBV LC-PCR assay may have been due in part to the automated DNA purification and sample handling on the MagNA Pure LC instrument, as variations in real-time PCR assays have mainly been attributed to variations in the nucleic acid purification procedures (24). In general, automation should result in more reliable and standardized test results by molecular assays of clinical samples. The results of the HBV LC-PCR assay were found to be in good agreement with those of the widely used, standardized Cobas Amplicor HBV Monitor test for the evaluation of clinical samples. All 100 samples with ⬎200 HBV DNA copies/ml by the Cobas Amplicor HBV Monitor Test also yielded ⬎200 HBV DNA copies/ml by the HBV LC-PCR assay. The quantification results of both assays were significantly correlated with each other. Although the detection limits of both assays were identical, i.e., 200 HBV DNA copies/ml, the results for samples with HBV DNA at levels below this limit were not in complete agreement, suggesting that the two assays have slightly different sensitivities. Testing of EU QCCA HBV Proficiency Panel 1999 by the HBV LC-PCR assay yielded results that ranged within 0.5 log10 of the target copy numbers for all panel members except panel member 2. This range was regarded as a good quantification result by the EU QCCA HBV program (32). As the previously described (6, 16, 28) HBV DNA-specific PCR assays with the LightCycler instrument did not test EU QCCA HBV panels, a comparison of our results with those of other HBV DNA-specific PCR assays with the LightCycler instrument was not possible. Participation in external quality control programs as well as the use of standardized reagents and materials is essential for comparison of the abilities of various assays to quantitate HBV DNA in patient samples (33). In conclusion, we have shown that the fully automated HBVLC PCR assay for the quantitative detection of HBV DNA described here exhibits a high degree of precision, a wide range of linearity, and a clinically relevant detection limit. The quantities of HBV DNA obtained by the assay correlated well with those obtained by the standardized Cobas Amplicor HBV Monitor test. The assay was provided with an IC to exclude false-negative results. The newly developed assay proved to be reliable, labor saving, and suitable for the routine clinical assessment of HBV DNA in plasma samples. ACKNOWLEDGMENT We are grateful to M. Nu ¨bling, Paul Ehrlich Institut, for the generous gift of the HBV reference material. REFERENCES 1. Abe, A., K. Inoue, T. Tanaka, J. Kato, N. Kajiyama, R. Kawaguchi, S. Tanaka, M. Yoshiba, and M. Kohara. 1999. Quantitation of hepatitis B virus genomic DNA by real-time detection PCR. J. Clin. Microbiol. 37:2899–2903. 2. Al-Soud, W. A., and P. Radstro ¨m. 2001. Purification and characterization of PCR-inhibitory components in blood cells. J. Clin. Microbiol. 39:485–493. 3. Alter, M. J. 2003. Epidemiology and prevention of hepatitis B. Semin. Liver Dis. 23:39–46. 4. Bai, X., B. B. Rogers, P. C. Harkins, J. Somerauer, R. Squires, K. Rotondo, A. Quan, D. B. Dawson, and R. H. Scheuermann. 2000. Predictive value of quantitative PCR-based viral burden analysis for eight human herpes viruses in pediatric sold organ transplant patients. J. Mol. Diagn. 2:191–201. 5. Beyer, A., J. Barner, H. W. Doerr, and B. Weber. 1998. Quantitation of viral load: clinical relevance for human immunodeficiency virus, hepatitis B virus and hepatitis C virus infection. Intervirology 41:24–34. 6. Brechtbuehl, K., S. A. Walley, G. M. Dusheiko, and N. A. Saunders. 2001. A rapid real-time quantitative polymerase chain reaction for hepatitis B virus. J. Virol. Methods 93:105–113.

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7. Chen, R. W., H. Piparinen, M. Seppanen, P. Koskela, S. Sarna, and M. Lappalainen. 2001. Real-time PCR for detection and quantitation of hepatitis B virus DNA. J. Med. Virol. 65:250–256. 8. DiDomenico, N., H. Link, R. Knobel, T. Caratsch, W. Weschler, Z. G. Loewy, and M. Rosenstraus. 1996. COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine diagnostic PCR. Clin. Chem. 42:1915–1923. 9. Elenitoba-Johnson, K. S., S. D. Bohling, C. T. Wittwer, and T. C. King. 2001. Multiplex PCR by multicolour fluorimetry and fluorescence melting curve analysis. Nat. Med. 7:249–253. 10. Espy, M. J., P. N. Rys, A. D. Wold, J. R. Uhl, L. M. Sloan, G. D. Jenkins, D. M. Ilstrup, F. R. Cockerill III, R. Patel, J. E. Rosenblatt, and T. F. Smith. 2001. Detection of herpes simplex virus DNA in genital and dermal specimens by LightCyler PCR after extraction using the IsoQuick, MagNA Pure, and BioRobot 9604 Methods. J. Clin. Microbiol. 39:2233–2236. 11. Fiebelkorn, K. R., B. G. Lee, C. E. Hill, A. M. Caliendo, and F. S. Nolte. 2002. Clinical evaluation of an automated nucleic acid isolation system. Clin. Chem. 48:1613–1615. 12. Gobbers, E., T. A. M. Oosterlaken, M. J. A. W. M. van Bussel, R. Melsert, A. C. M. Kroes, and E. C. J. Claas. 2001. Efficient extraction of virus DNA by NucliSens Extractor allows sensitive detection of hepatitis B virus by PCR. J. Clin. Microbiol. 39:4339–4343. 13. Hendricks, D. A., B. J. Stowe, B. S. Hoo, J. Kolberg, B. D. Irvine, P. D. Neuwald, M. S. Urdea, and P. R. Perrrillo. 1995. Quantitation of HBV DNA in human serum using branched DNA signal amplification assay. Am. J. Clin. Pathol. 104:537–546. 14. Ho, S. K. N., and T. M. Chan. 2000. An overview of assays for serum HBV DNA. Clin. Lab. 46:609–614. 15. Jardi, R., M. Buti, F. Rodriguez-Frias, X. Costa, M. Cortina, R. Esteban, J. Guardia, and C. Pascual. 1996. The value of quantitative detection of HBVDNA amplified by PCR in the study of hepatitis B infection. J. Hepatol. 24:680–685. 16. Jardi, R., F. Rodriguez, M. Buti, X. Costa, M. Cortina, A. Valdez, R. Galimany, R. Esteban, and J. Guardia. 2001. Quantitative detection of hepatitis B virus DNA in serum by a new rapid real-time fluorescence PCR assay. J. Viral Hepatol. 8:465–471. 17. Kapke, G., G. Watson, S. Sheffler, D. Hunt, and C. Frederick. 1997. Comparison of the Chiron quantiplex branched DNA (bDNA) assay and the Abbott Genostics solution hybridization assay for quantitation of hepatitis B viral DNA. J. Viral Hepatol. 4:67–75. 18. Kessler, H. H. 2000. Semiautomated quantification of hepatitis B virus DNA in a routine diagnostic laboratory. Clin. Diagn. Virol. 7:853–855. 19. Kessler, H. H., S. Preininger, E. Stelzl, E. Daghofer, B. I. Santner, E. Marth, H. Lackner, and R. Stauber. 2000. Identification of different states of hepatitis B virus infection with a quantitative PCR assay. Clin. Diagn. Lab. Immunol. 7:298–300. 20. Kessler, H. H., G. Mu ¨hlbauer, E. Stelzl, E. Daghofer, B. I. Santner, and E. Marth. 2001. Fully automated nucleic acid extraction: MagNA Pure LC. Clin. Chem. 47:1124–1126. 21. Krajden, M., J. Minor, L. Cork, and L. Comanor. 1998. Multi-measurement

J. CLIN. MICROBIOL.

22. 23. 24.

25. 26. 27. 28.

29.

30. 31. 32.

33. 34.

35. 36.

method comparison of three commercial hepatitis B virus DNA quantification assays. J. Viral Hepatol. 5:415–422. Lai, V. C. H., R. Guan, M. L. Wood, S. K. Lo, M. F. Yuen, and C. L. Lai. 1999. Nucleic acid based cross-linking assay for the detection and quantification of hepatitis B virus DNA. J. Clin. Microbiol. 37:161–164. Loeb, K. R., K. R. Jerome, J. Goddard, M. Huang, A. Cent, and L. Corey. 2000. High-throughput quantitative analysis of hepatitis B virus DNA in serum using TaqMan fluorogenic detection system. Hepatology 32:626–629. Lo ¨ffler, J., K. Schmidt, H. Hebart, U. Schumacher, and H. Einsele. 2002. Automated extraction of genomic DNA from medically important yeast species and filamentous fungi by using the MagNA Pure LC system. J. Clin. Microbiol. 40:2240–2243. Lok, A. S. F. 1994. Treatment of chronic hepatitis B. J. Viral Hepatol. 1:105–124. Lopez, V. A., E. J. Bourne, M. W. Lutz, and L. D. Condreay. 2002. Assessment of the Cobas Amplicor HBV Monitor test for the quantitation of serum hepatitis B virus DNA levels. J. Clin. Microbiol. 40:1972–1976. Noborg, U., A. Gusdal, E. K. Pisa, A. Hedrum, and M. Lindh. 1999. Automated quantitative analysis of hepatitis B virus DNA by using the Cobas Amplicor HBV Monitor test. J. Clin. Microbiol. 37:2793–2797. Paraskevis, D., C. Haida, N. Tassopoulos, M. Ratopoulou, D. Tsantoulas, H. Papachristou, V. Sypsa, and A. Hatzakis. 2002. Development and assessment of a novel real-time PCR assay for quantitation of HBV DNA. J. Virol. Methods 103:201–212. Pas, S. D., E. Fries, R. A. de Man, A. D. M. E. Osterhaus, and H. G. M. Niesters. 2000. Development of a quantitative real-time detection assay for the hepatitis B virus DNA and comparision with two commercial assays. J. Clin. Microbiol. 38:2897–2901. Sto ¨cher, M., V. Leb, G. Ho ¨lzl, and J. Berg. 2002. A simple approach to the generation of heterologous competitive internal controls for real-time PCR assays on the LightCycler. J. Clin. Virol. 25:S47–S53. Sto ¨cher, M., and J. Berg. 2002. Normalized quantification of human cytomegalovirus DNA by competitive real-time PCR on the LightCycler instrument. J. Clin. Microbiol. 40:4547–4553. Valentine-Thon, E., A. M. van Loon, J. Schirm, J. Reid, P. E. Klapper, and G. M. Cleator. 2001. European proficiency testing program for molecular detection and quantitation of hepatitis B virus DNA. J. Clin. Microbiol. 39:4407–4412. Valentine-Thon, E. 2002. Quality control in nucleic acid testing—where do we stand? J. Clin. Virol. 25:S13–S21. Van Deursen, F. J., K. Hino, D. Wyatt, P. Molyneaux, P. Yates, L. A. Wallace, B. C. Dow, and W. F. Carman. 1998. Use of PCR in resolving diagnostic difficulties potentially caused by variation of hepatitis B virus. J. Clin. Pathol. 51:149–153. Wittwer, C. T., M. G. Herrmann, A. A. Moss, and R. P. Rasmussen. 1997. Continuous fluorescence monitoring of rapid cycle DNA amplification. BioTechniques 22:130–138. Wittwer, C. T., M. K. Ririe, R. V. Andrew, D. A. David, R. A. Gundry, and U. J. Balis. 1997. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. BioTechniques 22:176–181.