Ligase chain reaction (LCR®) assay for semi‐quantitative detection of HBV DNA in mononuclear leukocytes of patients with chronic hepatitis B

Trippler, Martin; Hampl, H.; Goergen, Bernd; Spies, U.; Knolle, Percy A.; Grimm, B; Büschenfelde, K-H. Meyerzum; Gerken, Guido

doi:10.1111/j.1365-2893.1996.tb00054.x

Cited by 9 publications

(4 citation statements)

References 23 publications

(2 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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“…On the contrary, the frequency of anti-HCV antibodies among NHL mirrors the local prevalence in the general population [17,21,33,34], as if the lymphoma patients may act as a reservoir for a virus, common and easily growing in the lymphoid tissue independently of its oncogenic power. This hypothesis is supported by the prevalence of infection from HBV, higher than in the control group and possibly more than HCV prevalence itself, and by the observation that also the HBV has a tropism to mononuclear blood cells [35][36][37][38][39]. Occasional patients suffering from chronic hepatitis have been reported to develop NHL with exclusive liver involvement [19,40,41] and some series of patients with HCV-related liver disease have been reported to have a greater risk of developing NHL [42].…”

Section: Discussionmentioning

confidence: 88%

Hepatitis C virus in non-Hodgkin's lymphoma. A reappraisal after a prospective case-control study of 300 patients

et al. 2000

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It is widely thought, but not yet explained, that there might be a pathogenetic link between the infection of hepatitis C virus (HCV) and the onset of B non-Hodgkin's lymphoma (NHL). We studied the prevalence of serum anti-HCV antibodies among 300 NHL comparing it with the prevalence among 600 age- and sex-matched non-neoplastic subjects as controls, 247 patients with non-lymphomatous neoplasm, and 122 patients treated with immunosuppressive agents. We found a prevalence of 0.16 among NHL and 0.085 among controls and non-lymphomatous patients. Although the difference was statistically significant (P < 0.001), the odds ratio was 2.049 and its confidence intervals included the equality. The HCV prevalence was independent of NHL subset, and the genotypes distribution was the same among NHL and controls. We disclosed a HBsAg prevalence of 0.077 in NHL versus 0.008 in controls (P < 0.001) with an odds ratio of 9.9. We do not believe that these findings support the hypothesis of an HCV pathogenetic role in lymphomagenesis because (i) the risk of previous infection is marginally higher in NHL than in controls, (ii) a typical genotype distribution is lacking, as is a NHL clinico-histological feature associated with HCV, and (iii) the higher prevalence of viral infection is not specific as witnessed by the high HBsAg prevalence.

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“…The potential drawbacks of LCR include increased background signal and false positive results when target independent ligation occurs in the absence of template DNA, difficulty in inactivating post-amplification products, and the inability to incorporate effective contamination control methods [100,101]. LCR assays have been successfully developed for the detection of several viruses such as HPV [102], HSV 1 & 2 [103], and HBV [104]; however, no commercial LCR-based viral assays are currently available in the U.S.…”

Section: Ligase Chain Reaction (Lcr)mentioning

confidence: 99%

Amplification chemistries in clinical virology

Dunbar

Das

2019

Journal of Clinical Virology

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Molecular diagnostic methods have evolved and matured considerably over the last several decades and are constantly being evaluated and adopted by clinical laboratories for the identification of infectious pathogens. Advancement in other technologies such as fluorescence, electronics, instrumentation, automation, and sensors have made the overall diagnostic process more accurate, sensitive, and rapid. Nucleic acid based detection procedures, which rely on the fundamental principles of DNA replication have emerged as a popular and standard diagnostic method, and several commercial assays are currently available based on different nucleic acid amplification techniques. This review focuses on the major amplification chemistries that are used for developing commercial assays and discusses their application in the clinical virology laboratory. Polymerase chain reaction (PCR)The development of PCR by Kary Mullis in the 1980s revolutionized the detection of microbial pathogens in clinical specimens. PCR is a highly sensitive method that involves cyclical heating and cooling of the reaction mixture to facilitate amplification of the target DNA [3]. The method consists of three basic steps (denaturation, annealing, and extension) and the reaction mixture requires four primary components (template DNA, primers, nucleotides, and a DNA polymerase) (Fig. 1a). Double-stranded target DNA (dsDNA) is thermally denatured to form a single-stranded DNA template (ssDNA). Oligonucleotide primers anneal to complementary target sequences on the nucleic acid template and are extended by the DNA polymerase, thus creating the resultant PCR product (amplicon). The reaction occurs in a programmable thermal https://doi.

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Ligase chain reaction (LCR®) assay for semi‐quantitative detection of HBV DNA in mononuclear leukocytes of patients with chronic hepatitis B

Cited by 9 publications

References 23 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

Hepatitis C virus in non-Hodgkin's lymphoma. A reappraisal after a prospective case-control study of 300 patients

Amplification chemistries in clinical virology

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