Real time TaqMan PCR detection and quantitation of HBV genotypes A–G with the use of an internal quantitation standard

Weiss, Judith B.; Wu, Hui; Farrenkopf, B.; Schultz, T.; Song, Geun Am; Shah, Syed İnayat Ali; Siegel, Jeffrey

doi:10.1016/j.jcv.2003.08.015

Cited by 72 publications

(76 citation statements)

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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.…”

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confidence: 99%

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confidence: 99%

“…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.…”

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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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“…In the last few years, real-time PCR methods with wider detection ranges have been reported (1-4, 8, 10, 12, 13, 16, 17, 19-24). Here we evaluate the detection range, reproducibility, and clinical applicability of the Cobas Taqman HBV assay (Roche Molecular Systems, Branchburg, NJ), a real-time PCR method which includes an internal quantitation standard (23).…”

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Dynamic Range and Reproducibility of Hepatitis B Virus (HBV) DNA Detection and Quantification by Cobas Taqman HBV, a Real-Time Semiautomated Assay

Lindh

Hannoun

2005

J Clin Microbiol

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The Cobas Taqman assay for hepatitis B virus (HBV) DNA showed linear detection over 7 logs for genotypes A to D. The coefficient of variation was 1.2% at >1,000 IU/ml and 22.0% at 10 IU/ml. In 97 clinical samples, the log HBV DNA/ml differed by 0.11 between Cobas Amplicor and Cobas Taqman (r 2 ‫؍‬ 0.97).Hepatitis B virus (HBV) infection is a major cause of chronic liver disease and may result in liver cirrhosis or hepatocellular carcinoma. In chronic infection, viremia in general persists lifelong but at highly variable levels, from more than 10 9 copies/ml to below 100 copies/ml. Quantitation of HBV DNA is important for staging and pretreatment evaluation (5,6,11,15). Moreover, antiviral therapy requires highly sensitive detection of viremia, both for monitoring the initial response and later for identifying increasing HBV DNA levels indicating drug resistance (10,12,14).Methods for analyzing HBV DNA by quantitative PCR have had limitations, mainly concerning the detection range (7,9,18). In the last few years, real-time PCR methods with wider detection ranges have been reported (1-4, 8, 10, 12, 13, 16, 17, 19-24). Here we evaluate the detection range, reproducibility, and clinical applicability of the Cobas Taqman HBV assay (Roche Molecular Systems, Branchburg, NJ), a real-time PCR method which includes an internal quantitation standard (23).The analyses were performed with a Cobas Taqman 48 instrument according to the manufacturer's instructions. First, HBV DNA was manually isolated from 500 l serum. A known number of quantitation standard (QS) molecules were introduced into each specimen and were carried through the specimen preparation, amplification, and detection steps, serving as both a quantitation standard and an inhibition control. The DNA was eluted in a volume of about 80 l, of which 50 l was used for PCR in a reaction mixture of 100 l, amplifying a 105-bp segment of the precore-core region. The C T values, i.e., the cycles in which the fluorescence becomes detectable for target HBV and QS, are used to calculate the target HBV concentration, which essentially equals to [QS](2 ⌬C T ), where [QS] represents the concentration of added QS and ⌬C T is the difference in C T for HBV and QS.Linearity panels consisting of samples (genotypes A, B, C, and D) with high HBV DNA levels (Ͼ10 9 copies/ml as measured by Cobas Amplicor [Roche Diagnostics, Branchburg, NJ]) were prediluted to approximate levels of 10 8 copies/ml and serially diluted in 1:10 steps to around 10 2 copies/ml. At each level, two replicates were analyzed by Cobas Taqman (DNA extraction and real-time PCR). As shown in Fig. 1, there was a good linearity over 7 logs for all genotypes (A to D). The r 2 values were 0.997, 0.997, 0.997, and 0.991 for genotypes A, B, C, and D.Reproducibility was evaluated by analyzing a genotype D sample that was diluted to six replicates at four different levels (5 ϫ 10 7 , 5 ϫ 10 5 , 5 ϫ 10 3 , and 50 copies/ml). Each of the 24 replicates was analyzed by Cobas Taqman (DNA extraction and real-time PCR) on 4 subs...

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Real time TaqMan PCR detection and quantitation of HBV genotypes A–G with the use of an internal quantitation standard

Cited by 72 publications

References 23 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

Dynamic Range and Reproducibility of Hepatitis B Virus (HBV) DNA Detection and Quantification by Cobas Taqman HBV, a Real-Time Semiautomated Assay

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