Fully automated quantitation of Hepatitis B virus (HBV) DNA in human plasma by the COBAS® AmpliPrep/COBAS® TaqMan® System☆, ☆☆

Hochberger, S.; Althof, D.; Schrott, R. Gallegos de; Nachbaur, N.; Röck, H.; Leying, Hermann

doi:10.1016/j.jcv.2006.01.003

Cited by 59 publications

(53 citation statements)

References 31 publications

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“…Comparison of the two assays for quantitative detection of HBV DNA levels in the 118 serum samples showed that the two sets of results were highly correlated (r = 0.948, P < 0.001). This was in accordance with the results of previous studies that have evaluated other realtime PCR assays for quantitation of HBV DNA [13,14,[22][23][24][25][26] .…”

Section: Discussionsupporting

confidence: 92%

Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

Shi¹,

Zhang²,

Zhu³

et al. 2008

WJG

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INTRODUCTIONIt is estimated that 350 million individuals are chronically infected with hepatitis B virus (HBV) worldwide, and about one-third of these live in China [1] . HBV infection can cause chronic hepatitis, cirrhosis and hepatocellular carcinoma [2] . Among patients with active viral replication, cirrhosis will develop in 15%-20% within 5 year [3] , and 70-90 percent of cases of hepatocellular carcinoma occur against a background of cirrhosis [4] . Quantitation of HBV DNA in serum or plasma has been an important tool in identifying individuals with active hepatitis B, to monitor the efficiency of antiviral treatment, and to predict whether the treatment will be successful [5][6][7][8][9] .Several commercial molecular assays have been developed for quantitation of HBV DNA in serum or plasma samples. One of these assays is the COBAS Amplicor HBV Monitor, which is based on the amplification of DNA targets by the use of HBV-specific primers. Other methods, such as the VERSANT HBV DNA 3.0 assay, which is based on branched-DNA signal amplification, and Hybrid Capture Ⅱ, which is based on hybridization of a chemiluminescent probe, are also routinely used in diagnostic laboratories [10,11] . However, the costs of these assays are too high to be used in developing countries such as China, and in these countries the HBV burden is much heavier than in developed countries.Recently, quantitative real-time PCR has been used to detect and quantify HBV levels in serum or plasma samples. This assay is based on the relationship between the initial DNA target levels and the threshold cycle (Ct) of the amplification products. The Ct value is smaller when the initial DNA target level is higher. Several evaluation studies have shown that real-time PCR has a higher sensitivity, a broader dynamic range, and an accurate quantitation of HBV DNA compared to those of the existing commercial methods [12][13][14][15] .The Fosun real-time PCR HBV kit is a commercial METHODS:The reference sera of the Chinese National Institute for the Control of Pharmaceutical and Biological Products and the National Center for Clinical Laboratories of China, and 158 clinical serum samples were used in this study. The linearity, accuracy, reproducibility, assay time, and costs of the real-time PCR were evaluated and compared with those of the Cobas Amplicor test. RESULTS:The intra-assay and inter-assay variations of the real-time PCR ranged from 0.3% to 3.8% and 1.4% to 8.1%, respectively. The HBV DNA levels measured by the real-time PCR correlated very well with those obtained with the COBAS Amplicor test (r = 0.948). The real-time PCR HBV DNA kit was much cheaper and had a wider dynamic range. CONCLUSION:The real-time PCR assay is an excellent tool for monitoring of HBV DNA levels in patients with chronic hepatitis B.

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Section: Discussionsupporting

confidence: 92%

Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

Shi¹,

Zhang²,

Zhu³

et al. 2008

WJG

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“…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.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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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“…In the presence of target, probe hybridization and primer extension occur simultaneously. The fluorescent signal is generated by removing the reporter through the 5Ј33Ј exonuclease activity of a thermostable Taq DNA polymerase (4,5). In the TaqMan and Abbott RealTime PCR assays an additional probe is used to detect genotype G or C, respectively.…”

Section: The New Abbott Realtime Hepatitis B Virus (Hbv) Assay Was Comentioning

confidence: 99%

“…The Cobas AmpliPrep extracts HBV-DNA from 850 l of serum, and during the extraction a known number of HBV quantitation standard DNA molecules are introduced in each specimen (5). The quantitation of HBV-DNA is performed using the HBV quantitation standard.…”

Section: The New Abbott Realtime Hepatitis B Virus (Hbv) Assay Was Comentioning

confidence: 99%

Evaluation of the Abbott RealTime HBV DNA Assay and Comparison to the Cobas AmpliPrep/Cobas TaqMan 48 Assay in Monitoring Patients with Chronic Cases of Hepatitis B

et al. 2008

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The new Abbott RealTime hepatitis B virus (HBV) assay was compared to the Cobas AmpliPrep/CobasTaqMan assay with 128 serum samples from patients with chronic hepatitis B. There was an excellent correlation (r ‫؍‬ 0.961) between the two assays, with the Abbott RealTime test showing at least equivalent sensitivity and a slightly wider dynamic range than the Cobas TaqMan assay. By coupling high sensitivity with a large dynamic range, the Abbott RealTime HBV assay is useful in monitoring the response to antiviral therapy.Hepatitis B virus (HBV) is a small partially double-stranded virus belonging to the family of the Hepadnaviridae that can cause acute or chronic hepatitis (2). Millions of people around the world are infected by HBV, and it is estimated that about 350 million people worldwide have developed a persistent infection (6). Measurement of HBV levels in serum is a reliable marker for prognosis of acute and chronic infection and plays an important role in the management of patients receiving antiviral drugs (7,8). Therefore, diagnostic assays for the accurate quantitation of HBV DNA are essential for optimal patient management. Monitoring of HBV DNA allows to predict the success of antiviral therapy and to identify the development of drug resistance. Real-time PCR is a new molecular tool that is progressively replacing endpoint PCR systems for monitoring patients with chronic hepatitis B since it allows for high sensitivity, a broad dynamic range, accuracy, and rapid results (1, 9).Quantification by real-time PCR is based on the determination of the threshold cycle (C T ) when the amplified product is detected for the first time (4) and the PCR is still in the exponential phase. In this case the quantification of the viral load is much more accurate and reliable than that measured with endpoint PCR systems (3).The aim of our study was to evaluate the Abbott RealTime HBV assay for the detection and quantification of HBV-DNA in serum samples and to compare these results with those obtained using the Cobas AmpliPrep/Cobas TaqMan HBV test (Roche Diagnostics). The study was carried out on 128 serum samples collected from patients with chronic HBV infections attending our hepatology clinic at the University Hospital Tor Vergata, Rome, Italy.Blood samples were collected in a BD Vacutainer tube containing a separation gel. After centrifugation, the serum was divided into two aliquots of 1 ml each and tested by the Abbott and Cobas TaqMan Real-time PCR assays, respectively. Both systems use a hybridization probe with a fluorescent moiety covalently linked to the 5Ј end of the probe (reporter) and a quenching moiety bound to the 3Ј end of the probe (quencher). In the presence of target, probe hybridization and primer extension occur simultaneously. The fluorescent signal is generated by removing the reporter through the 5Ј33Ј exonuclease activity of a thermostable Taq DNA polymerase (4, 5). In the TaqMan and Abbott RealTime PCR assays an additional probe is used to detect genotype G or C, respectively. While the Coba...

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Fully automated quantitation of Hepatitis B virus (HBV) DNA in human plasma by the COBAS® AmpliPrep/COBAS® TaqMan® System☆, ☆☆

Cited by 59 publications

References 31 publications

Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

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 Abbott RealTime HBV DNA Assay and Comparison to the Cobas AmpliPrep/Cobas TaqMan 48 Assay in Monitoring Patients with Chronic Cases of Hepatitis B

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