Fully Automated, Internally Controlled Quantification of Hepatitis B Virus DNA by Real-Time PCR by Use of the MagNA Pure LC and LightCycler Instruments

Leb, Victoria; Stöcher, Markus; Valentine-Thon, Elizabeth; Hölzl, Gabriele; Kessler, Harald H.; Stekel, Herbert; Berg, Jörg

doi:10.1128/jcm.42.2.585-590.2004

Cited by 37 publications

(30 citation statements)

References 36 publications

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“…Real-time PCR assays for HBV DNA based on the TaqMan technology, as well as on other real-time platforms, have been designed on manual, semiautomatic, or automatic nucleic acid extraction procedures suitable for small series. Thus far, these formats have shown an excellent correlation with conventional endpoint PCR systems, including CAHBM, which is the most commonly used assay in comparative evaluations (7,15,18,21,24,31,32).…”

Section: Discussionmentioning

confidence: 99%

“…The real-time PCR system COBAS TaqMan 48 for HBV DNA has been evaluated with both automatic and manual extraction platforms, i.e., the MagNA Pure technology for small routine series and the generic High-Pure system viral nucleic acid extraction kit respectively, and has proved to be very reproducible and sensitive (9,12,18,35). The integration of the real-time PCR analyzer with a fully automated DNA extraction platform on the COBAS AmpliPrep instrument (CAP-CTM) for large routine series has been recently introduced.…”

Section: Discussionmentioning

confidence: 99%

“…The recently introduced real-time PCR technique represents the method of choice compared to previous, conventional endpoint PCR due to a very sensitive quantification of the viral load over a wide dynamic range (7,12,15,18,20,21,24,31,32,35,41). At present, sample preparation is a major weakness in molecular tests, and improvements are constantly introduced to decrease the variability of the techniques and the risk of contamination, such as ready-to-use reagents and automation of the extraction procedure.…”

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confidence: 99%

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COBAS AmpliPrep-COBAS TaqMan Hepatitis B Virus (HBV) Test: a Novel Automated Real-Time PCR Assay for Quantification of HBV DNA in Plasma

et al. 2007

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Hepatitis B virus (HBV) infection is a major cause of chronic liver disease. It is estimated that nearly 2 billion people are infected worldwide by HBV and that more than 350 million have persistent and chronic infection (38). HBV carriers have a high risk of developing long-term sequelae of hepatitis B, including cirrhosis and hepatocellular carcinoma that, in countries such as Italy with a moderate prevalence of HBV infection, account for nearly 25% of the indications for liver transplantation in reference centers (29). Recent advances in antiviral therapy, based on the development of new and more powerful nucleos(t)ide analogues, have dramatically improved chronic hepatitis B management, including the prevention of allograft reinfection in those patients undergoing liver transplantation for HBV-related disease.The success of antiviral therapy has been supported by the introduction of highly sensitive tests for monitoring HBV DNA. Molecular tests help to determine the activity of HBV infection, the selection of patients for treatment, and the efficacy of antiviral therapy, identifying the development of HBV drug-resistant strains (16,36,38).Several assays based on PCR are currently commercially available for HBV DNA. The recently introduced real-time PCR technique represents the method of choice compared to previous, conventional endpoint PCR due to a very sensitive quantification of the viral load over a wide dynamic range (7,12,15,18,20,21,24,31,32,35,41). At present, sample preparation is a major weakness in molecular tests, and improvements are constantly introduced to decrease the variability of the techniques and the risk of contamination, such as ready-to-use reagents and automation of the extraction procedure.A fully automated system, the COBAS AmpliPrep-COBAS TaqMan HBV test (CAP-CTM; Roche Molecular Systems, Inc., Branchburg, NJ) consisting of two integrated platformsthe COBAS AmpliPrep for automated nucleic acid extraction from plasma specimens and the COBAS TaqMan 48, a realtime PCR assay based on TaqMan technology-has recently been developed (13,35). Important improvements compared to other real-time PCR assays for HBV DNA are the incorporation of an internal quantitation standard to monitor the efficiency of the entire process and the introduction of a system to prevent carryover contamination. CAP-CTM is suitable for large routine series and has been demonstrated to equally quantitate HBV genotypes A through G (13, 35), but at present there are only few clinical data (13,28).In the present study, the performance of the CAP-CTM was evaluated and compared to the endpoint PCR assay COBAS AMPLICOR HBV monitor (CAHBM; Roche Molecular Systems). Analytical sensitivity and precision were assessed with an HBV proficiency panel, while correlation and differences in * Corresponding author. Mailing address: Laboratory of Microbiology, Molinette Hospital, Corso Bramante 88/90,

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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COBAS AmpliPrep-COBAS TaqMan Hepatitis B Virus (HBV) Test: a Novel Automated Real-Time PCR Assay for Quantification of HBV DNA in Plasma

et al. 2007

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“…Due to an increasing demand for molecular tests and for taking advantage of real-time PCR, a fully automated extraction system is considered a prerequisite (18,19,36). Several solutions for nucleic acid (NA) extraction on automated platforms have recently been evaluated for the diagnosis of viral infections (9,11,15,17,20,22,26,27). The COBAS AmpliPrep system (CAP; Roche Diagnostics) is a new instrument designed for rapid high-throughput purification of DNAs and RNAs from biological fluids (such as serum and plasma) (5,14,19,31,33,34).…”

mentioning

confidence: 99%

Evaluation of the COBAS AmpliPrep-Total Nucleic Acid Isolation-COBAS TaqMan Hepatitis B Virus (HBV) Quantitative Test and Comparison to the VERSANT HBV DNA 3.0 Assay

Ronsin¹,

Pillet²,

Bali³

et al. 2006

J Clin Microbiol

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Quantitative detection of hepatitis B virus (HBV) in serum or plasma has become the most direct and reliable method for monitoring chronic hepatitis B. Here, we report the performance characteristics of a real-time PCR hepatitis B DNA quantitative assay, the COBAS TaqMan (CTM) HBV test (Roche Diagnostics, Meylan, France), in combination with an automated DNA extraction on the COBAS AmpliPrep (CAP) instrument using the total nucleic acid isolation kit (TNAI kit), a generic reagent for nucleic acid isolation (both from Roche Diagnostics). The linearity, accuracy, and specificity of the CAP-TNAI-CTM HBV test were evaluated using various reference panels and standards (HBV panel 2004 from Quality Control for Molecular Diagnostics, OptiQuant HBV panel from AcroMetrix, WHO International Standard for HBV, and Teragenix hepatitis B genotype panel). Quantitative results show that the CAP-TNAI-CTM HBV test performed well with respect to linearity, accuracy, and reproducibility from at least 100 to 500,000 HBV DNA IU/ml. Based on the log 10 IU of HBV DNA/ml measured, the intra-assay variation ranged from 2.49% to 8.46% and the interassay variation ranged from 1.88% to 7.83%. The test was extremely sensitive and could detect samples containing HBV DNA below the reported quantification threshold (<30 IU/ml). All HBV genotypes were correctly amplified, and no cross-contamination occurred during the automated sample preparation. In addition, 402 human serum samples were tested comparatively to the VERSANT HBV DNA 3.0 assay (bDNA; Bayer Diagnostics, Puteaux, France). The viral load results of the CAP-TNAI-CTM test and bDNA were significantly correlated, but the agreement between the two tests was poor, with large differences between results for individual samples. The hands-on time was estimated to be reduced from 2.30 h with bDNA to 45 min with the CAP-TNAI-CTM test, and up to 84 samples were completely processed within a working day. Overall, the performance characteristics of the CAP-TNAI-CTM test demonstrated that it provides a high-throughput sensitive and reliable method for quantitation of HBV DNA levels in the routine molecular laboratory.Hepatitis B virus (HBV) is a major causative agent of chronic hepatitis. Approximately 350 million to 400 million people worldwide are chronically infected. Chronic HBV infection can lead to liver cirrhosis and hepatocellular carcinoma (13). Detection and quantification of HBV DNA in serum or plasma are now considered important indicators for managing disease in HBVinfected patients and predicting and monitoring the efficiency of antiviral treatment as well as identifying the emergence of drug resistance by detecting HBV DNA breakthrough (4, 24). Several commercial molecular tests for the quantitation of HBV DNA in serum or plasma have been developed and are routinely used in diagnostic virology laboratories (10, 29). These molecular tests are based on target amplification (HBV Monitor test; Roche Diagnostics, Meylan, France), branched-DNA signal amplification (VERSANT HBV DNA 3.0 assa...

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“…13 internal Control Clinical specimens may contain inhibitors of PCR amplification, which can be monitored by using internal controls (IC). 20 These controls can be heterologous PCR systems or modified target variants, for example those containing a mutated probe-binding site. DNA based controls monitor the performance of the DNA PCR only, whereas RNA based controls enable to examine in addition the extraction of the sample and the reverse transcription step.…”

Section: Sequences Of Primers and Probesmentioning

confidence: 99%

Simultaneous Quantification and Genotyping of Hepatitis C Virus RNA by a Two-Step Real-Time PCR Assay on the Lightcycler Instrument

Abe

Klett²,

Wieland³

et al. 2010

Folia Medica

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Background: clinically, both viral load and genotypes have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis c and they are, under normal circumstances, performed as separate assays. design and methods: in order to improve the diagnostic strategy and subsequently reduce the reagent costs we have developed and established the simultaneous quantification and genotyping of hepatitis c virus rna by a two-step real-time pcr on the lightcycler instrument (roche diagnostics). results: The quantification assay was calibrated against WHO Standard 96/790. The detection limit was 30 IU/ml, the dynamic range up to 500,000,000 IU/ml. Intra-and interassay imprecisions were 1.2% and 1.9% (n = 10), respectively. The HCV RNA values obtained by real-time pcr assay were highly correlated with those obtained by the cobas Amplicor HCV monitor test (r = 0.992; p < 0.001). conclusions: the genotyping was performed by means of the melting temperature analysis. The concordance between our new genotyping method and the Trugene HCV 5'NC Kit was at the level of genotypes 100%. This rapid (3 h) and convenient assay is suitable for hcv genotyping, hcv detection and disease monitoring.

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Fully Automated, Internally Controlled Quantification of Hepatitis B Virus DNA by Real-Time PCR by Use of the MagNA Pure LC and LightCycler Instruments

Cited by 37 publications

References 36 publications

COBAS AmpliPrep-COBAS TaqMan Hepatitis B Virus (HBV) Test: a Novel Automated Real-Time PCR Assay for Quantification of HBV DNA in Plasma

COBAS AmpliPrep-COBAS TaqMan Hepatitis B Virus (HBV) Test: a Novel Automated Real-Time PCR Assay for Quantification of HBV DNA in Plasma

Evaluation of the COBAS AmpliPrep-Total Nucleic Acid Isolation-COBAS TaqMan Hepatitis B Virus (HBV) Quantitative Test and Comparison to the VERSANT HBV DNA 3.0 Assay

Simultaneous Quantification and Genotyping of Hepatitis C Virus RNA by a Two-Step Real-Time PCR Assay on the Lightcycler Instrument

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