Detection of hepatitis B virus in serum using amplification of viral DNA by means of the polymerase chain reaction

Sumazaki, Ryo; Motz, Manfred; Wolf, Hans; Heinig, Jutta; Jilg, Wolfgang; Deinhardt, Friedrich

doi:10.1002/jmv.1890270409

Cited by 84 publications

(38 citation statements)

References 22 publications

(8 reference statements)

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“…Several groups have reported the development of assays for the detection of HBV DNA with or without DNA amplification (1,8,17,21,26,34,37). The assays without amplification had lower detection sensitivities (from about 10 6 to 10 9 copies/ ml) and high quantitative accuracy, whereas the assays with amplification had higher sensitivities but lower quantitative FIG.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Detection of Hepatitis B Virus DNA by Real-Time Nucleic Acid Sequence-Based Amplification with Molecular Beacon Detection

Yates¹,

Penning²,

Goudsmit

et al. 2001

J Clin Microbiol

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We have developed a hepatitis B virus (HBV) DNA detection and quantification system based on amplification with nucleic acid sequence-based amplification (NASBA) technology and real-time detection with molecular beacon technology. NASBA is normally applied to amplify single-stranded target RNA, producing RNA amplicons. In this work we show that with modifications like primer design, sample extraction method, and template denaturation, the NASBA technique can be made suitable for DNA target amplification resulting in RNA amplicons. A major advantage of our assay is the one-tube, isothermal nature of the method, which allows high-throughput applications for nucleic acid detection. The homogeneous real-time detection allows a closed-tube format of the assay, avoiding any postamplification handling of amplified material and therefore minimizing the risk of contamination of subsequent reactions. The assay has a detection range of 10 3 to 10 9 HBV DNA copies/ml of plasma or serum (6 logs), with good reproducibility and precision. Compared with other HBV DNA assays, our assay provides good sensitivity, a wide dynamic range, and high-throughput applicability, making it a viable alternative to those based on other amplification or detection methods.Infection with hepatitis B virus (HBV) can cause a spectrum of liver diseases, such as fulminant or chronic hepatitis, cirrhosis, and hepatocellular carcinoma. About 300 million people throughout the world suffer from chronic HBV infection, and 2 billion people have markers indicating past infection with HBV (15,25).HBV is the smallest DNA virus known, and its genome shows a highly compact organization. A unique aspect in the HBV replication cycle is that a pregenomic mRNA serves as a template for the synthesis of the first viral DNA strand by the reverse transcriptase (RT) polymerase of HBV (35). The RNase H activity of the HBV DNA polymerase removes the mRNA during this process, and synthesis of the complementary second DNA strand is then started, generating a partially double-stranded DNA molecule for packaging in virions. When the virus enters the host, this molecule is extended into a fully double-stranded DNA molecule, thus starting a new replication cycle (10,27,28).HBV DNA can be detected in the blood in more than 90% of infected hosts who are positive for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). The use of detection and quantification of HBV DNA has become the preferred method for measuring the quantity of infectious particles and provides important diagnostic and prognostic information, mainly as a marker of virus replication (5,14,20,29,30).Numerous assays are available for detection of HBV DNA, such as the branched DNA (bDNA) assay (6, 13), DNA hybridization assays (9, 24), and quantitative PCR (1,12,22). Some of these assays have only limited sensitivity, however, and detection by PCR may be considered to be laborious and susceptible to contamination. Since HBV DNA loads are highly variable and require a broad dynamic detection range ...

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Section: Discussionmentioning

confidence: 99%

Quantitative Detection of Hepatitis B Virus DNA by Real-Time Nucleic Acid Sequence-Based Amplification with Molecular Beacon Detection

Yates¹,

Penning²,

Goudsmit

et al. 2001

J Clin Microbiol

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“…17 Total hepatic DNA was extracted using the Quiagen DNeasy Tissue Kit, and serum DNA was extracted using the QUIamp DNA Mini Kit (Quiagen Inc., Valencia, CA). A PCR product of 523 base pairs was generated in the first round of amplification using outer primers originally described by Sumazaki, 18 and an antisense primer 5Ј-TCGTCGTCTAACAACAGTAGTTTCCGG-3Ј was then used to amplify a seminested product of 421 base pairs. The PCR products were visualized on 0.8% agarose gel stained with ethidium bromide and processed in parallel with a water blank negative control, and DNA derived from HBsAg-positive serum and liver were used as a positive control.…”

Section: Methodsmentioning

confidence: 99%

Does lamivudine prophylaxis eradicate persistent HBV DNA from allografts derived from anti-HBc-positive donors?

Loss

Mason

Nair

et al. 2003

Liver Transplantation

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“…[25][26][27] As a control for HBV DNA contamination in the RNA samples, the 5Ј sense primer was used for the reversetranscription step in the two-step method instead of the 3Ј antisense primer, and, additionally, the reverse-transcription step was omitted before the PCR amplification. Also, as a specificity control for the one-step method, each sample was pretreated with RNAse A (Sigma) to ensure that a positive signal was abrogated by RNAse pretreatment and only HBV RNA was being amplified.…”

Section: Methodsmentioning

confidence: 99%

“…[25][26][27] Following high-stringency washes at 65°C for the full-length HBV-DNA probe, or at 42°C for the oligonucleotide probes, the filters were exposed to X Omat film (Kodak, Rochester, NY) for 24 hours to 1 week at Ϫ70°C.…”

Section: Methodsmentioning

confidence: 99%

Molecular basis for persistent hepatitis B virus infection in the liver after clearance of serum hepatitis B surface antigen

Mason¹,

Xu²,

Guo³

et al. 1998

Hepatology

194

140

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The clearance of the hepatitis B surface antigen (HBsAg) from serum usually indicates a resolution of biochemical and histological hepatitis in patients with chronic hepatitis B. 1 However, there are clearly documented cases of reactivation of latent viral infection following chemotherapy and immunosuppressive treatment, as well as de novo infection in patients receiving organs from HBsAg-negative donors with serological evidence of previous hepatitis B virus (HBV) infection. [2][3][4][5][6][7] In this setting, the reactivated infection is not entirely unexpected, because most patients who have cleared HBsAg from the serum still have detectable HBV DNA in the liver using the polymerase chain reaction (PCR) methodology. [8][9][10][11][12][13][14] In fact, HBV DNA can also be found in bodily secretions and peripheral blood mononuclear cells from patients with acute and chronic HBV infection after sustained loss of serum HBsAg. [15][16][17] Taken together, these studies suggest that following the loss of HBsAg from serum, HBV persists in a state of low-level replication or in a replication-competent state that can be reactivated to form infectious particles.The natural biology of the latent virus may be further investigated by assessing the transcriptional activity and the molecular form of the persistent HBV DNA. Using in situ hybridization, we were unable to detect hepatic HBV RNA in patients who had cleared serum HBsAg, and other investigators had little success using reverse-transcription (RT) PCR studies because of the inability to completely eradicate tissue HBV DNA. 11 Furthermore, the molecular form of the persistent HBV DNA has proved difficult to define because of the minute quantities of virus found in patients with resolved hepatitis.During chronic infection, HBV DNA can be detected in four predominant molecular forms. Following infection by the intact Dane particle, the partially double-stranded HBV-DNA genome becomes a covalently closed circular (CCC) DNA molecule that acts as a template for transcription of mRNA and the RNA pregenome. The reverse transcription of the RNA pregenome and second-strand HBV DNA synthesis results in low-molecular-weight replicative intermediates, whereas the HBV DNA that integrates into the host' s genome can be detected as high-molecular-weight species on Southern blot. 18,19 Similar to retroviral integration at the site of the long terminal repeats, HBV DNA generally inserts into DNA flanked by the direct repeat regions, which share sequence homology with the U5 region of murine leukemia virus long terminal repeats. [20][21][22] The genomic organization of the HBV direct repeat region provides a potential strategy for the detection of replicating virus when only minute quantities of HBV DNA are present (Fig. 1). Using PCR primers flanking the direct repeat region, the CCC HBV DNA can be amplified using PCR. In contrast, the HBV DNA within the Dane particle is incomplete, and integrated HBV DNA is usually, but not always, disrupted in the direct repeat region (Fig. 1). Th...

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Detection of hepatitis B virus in serum using amplification of viral DNA by means of the polymerase chain reaction

Cited by 84 publications

References 22 publications

Quantitative Detection of Hepatitis B Virus DNA by Real-Time Nucleic Acid Sequence-Based Amplification with Molecular Beacon Detection

Quantitative Detection of Hepatitis B Virus DNA by Real-Time Nucleic Acid Sequence-Based Amplification with Molecular Beacon Detection

Does lamivudine prophylaxis eradicate persistent HBV DNA from allografts derived from anti-HBc-positive donors?

Molecular basis for persistent hepatitis B virus infection in the liver after clearance of serum hepatitis B surface antigen

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