Detection of hepatitis B virus DNA by real-time PCR using TaqMan-MGB probe technology

Zhao, Jinrong; Bai, Yujie; Zhang, Qinghua; Wei, Yan; Ding, Li; Yan, Xiaojun

doi:10.3748/wjg.v11.i4.508

Cited by 37 publications

(17 citation statements)

References 8 publications

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“…The interassay and intra-assay percent coefficient of variation for the logarithmic viral load value ranged from 2.78% to 6.27% for HCV and from 1.85% to 8.11% for HBV. These results are in concordance with those reported for manual or automated extraction systems and a real-time HCV-PCR (4, 6) or HBV-PCR (1,15,19). Samples of HCV and HBV proficiency program panels were tested by the respective in-house PCR assays after automated extraction.…”

Section: Discussionsupporting

confidence: 80%

Automated Extraction of Viral-Pathogen RNA and DNA for High-Throughput Quantitative Real-Time PCR

2005

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The performance of the m1000 system (Abbott Laboratories, Illinois) as a front-end extraction system for high-throughput "in-house" quantitative real-time PCR assays was analyzed and compared to that of manual extraction of plasma and serum samples ( hepatitis C virus [HCV] and hepatitis B virus [HBV]) and EDTAblood samples (cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). Linearity of extraction was tested on dilution series of HCV and HBV reference materials. The correlation coefficient for standard curves based on repeated extraction runs was 0.97 ؎ 0.06 for HCV and 0.97 ؎ 0.03 for HBV, indicating a linear extraction from 100 to 1.0 ؋ 10 5 HCV IU/ml and from 100 to 1.0 ؋ 10 6 HBV IU/ml. Intra-and interrun variability was below 0.23 log 10 IU/ml for 2.98 to 5.28 log 10 HCV IU/ml and 2.70 to 5.20 log 10 HBV IU/ml. Correlation between automated and manual extraction was very good. For HCV, the correlation coefficient was 0.91 and the mean difference in viral load was 0.13 log 10 HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log 10 HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log 10 copies/ml. Accuracy was confirmed with a reference panel (QCMD, Glasgow, Scotland) for all four assays. No cross-contamination was observed when extracting strongly positive polyomavirus samples (8.10 log 10 copies/ml) interspersed with polyomavirus-negative samples. Automated extraction via the m1000 system offers a high reliability of extraction and resulted in a strong reduction of the required extraction hands-on time for high-throughput PCR compared to manual extraction protocols.Real-time thermocyclers have greatly decreased the amount of hands-on time in nucleic acid (NA)-based diagnostics for pathogens. The use of commercially available universal master mixes that contain all reagents except the pathogen-specific primer-probe combination has further decreased the number of pipetting steps and thus reduced labor and the possibility of errors.NA extraction has now become the most critical and laborintensive step in NA-based diagnostic assays. The overall sensitivity of the assay is determined by the NA yield, its purity, and the amount of sample equivalents that can be transferred into the amplification reaction. Conventional manual sample preparation methods are labor intensive and susceptible to contamination, handling variations, or errors (3, 9, 12).Since both the pathogen range and the number of different sample types are expanding and since multiplex downstream testing is becoming standard practice, there is a need for a generic extraction method (2, 5, 10). Ideally, the NA extraction procedure should yield pure NA from different pathogens and from a broad range of sample types.Recently, Abbott introduced m1000, an automated generic RNA and DNA extraction system using magnetic microparticle processing (17, 18). The m1000 system provides the necessary features for full automation of NA extraction of up to...

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Section: Discussionsupporting

confidence: 80%

Automated Extraction of Viral-Pathogen RNA and DNA for High-Throughput Quantitative Real-Time PCR

2005

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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.…”

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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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“…The newly developed technique of minor groove binder (MGB) probe for diagnosis of virus infection has additional advantages over the traditional TaqMan probes (Fernandez et al. 2002; Zhao et al. 2005; Lole and Arankalle 2006).…”

Section: Introductionmentioning

confidence: 99%

Rapid detection of H5 avian influenza virus by TaqMan-MGB real-time RT-PCR

Yan

et al. 2007

Lett Appl Microbiol

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Aims: Real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) assay based on a TaqMan‐minor groove binder (MGB) probe was developed for the rapid detection of avian influenza virus subtype H5. Methods and Results: Conserved regions in the haemagglutinin genes of avian influenza viruses subtype H5 served as targets for the primers and TaqMan‐MGB probe design. Concentrations of primers and probe were optimized to improve the sensitivity and specificity of the reactions. A plasmid containing the haemagglutinin gene was constructed and in vitro transcribed for a quantitative assay of copy numbers of the target gene. The results revealed that the optimal concentration of primers and probe was 640 and 480 nmol l−1, respectively. The threshold of 100 copies of target molecules could be detected. The linear range for detection was determined as 102 to 108 molecules in reaction. Conclusions: It took less than 3 h to complete the detection from viral RNA extraction, with good sensitivity and repeatability. Significance and Impact of the Study: Real‐time RT‐PCR assay with MGB probe was an effective means for quick and quantitative laboratory detection and monitoring of H5 avian influenza viruses.

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Detection of hepatitis B virus DNA by real-time PCR using TaqMan-MGB probe technology

Abstract: Real-time PCR based on TaqMan-MGB probe technology is an excellent method for detection of HBV DNA.

Cited by 37 publications

References 8 publications

Automated Extraction of Viral-Pathogen RNA and DNA for High-Throughput Quantitative Real-Time PCR

Automated Extraction of Viral-Pathogen RNA and DNA for High-Throughput Quantitative Real-Time PCR

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

Rapid detection of H5 avian influenza virus by TaqMan-MGB real-time RT-PCR

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