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Quantitation of hepatitis B virus DNA by real-time PCR using internal amplification control and dual TaqMan MGB probes
- Source :
- Journal of Virological Methods. 135:83-90
- Publication Year :
- 2006
- Publisher :
- Elsevier BV, 2006.
-
Abstract
- Hepatitis B virus (HBV) DNA quantitation is used extensively for monitoring of antiviral treatment of HBV infection. A real-time PCR assay was developed using a TaqMan minor groove binder probe and primers corresponding to HBV pre-core region for HBV DNA quantitation. A 228 bp fragment from this genomic region of HBV was cloned and serial dilutions of plasmid DNA were used as an external DNA standard. Comparison of the real-time PCR quantitation results from 35 clinical serum samples with those obtained by COBAS Amplicor HBV DNA monitor kit (Roche Diagnostics) revealed a significant correlation (r = 0.92) for all the samples. The assay showed wide dynamic linear range between 2.5 x 10(2) and 2.5 x 10(10) copies/ml serum. Sera from 25 healthy individuals tested negative indicating the high specificity of the assay. The median coefficients of variation for both intra- and inter-experimental variability were 4.9% and 10.6%, respectively, which indicated remarkable reproducibility. An internal amplification control (IC) was developed to detect the presence of PCR inhibitors in the samples to avoid false negative results. The IC had the same primer binding sites but different internal sequence and it competed with the virus-derived target. The optimum concentration of IC was found to be 100 copies/reaction. The assay was validated by testing serial dilutions of the WHO international HBV DNA standard. Since conserved regions were considered during primer and probe design, the assay should be applicable to all HBV genotypes. The real-time assay will be useful for monitoring HBV-infected patients in routine diagnostic laboratories and in clinical practice enabling analysis of a wide dynamic range of HBV DNA in a single, undiluted sample.
- Subjects :
- Hepatitis B virus
Serial dilution
Statistics as Topic
Biology
medicine.disease_cause
Polymerase Chain Reaction
Sensitivity and Specificity
law.invention
chemistry.chemical_compound
law
Virology
TaqMan
medicine
Humans
Polymerase chain reaction
DNA Primers
Analysis of Variance
Reference Standards
Hepatitis B
biology.organism_classification
Molecular biology
Real-time polymerase chain reaction
chemistry
Hepadnaviridae
DNA, Viral
Reagent Kits, Diagnostic
Primer (molecular biology)
DNA
Subjects
Details
- ISSN :
- 01660934
- Volume :
- 135
- Database :
- OpenAIRE
- Journal :
- Journal of Virological Methods
- Accession number :
- edsair.doi.dedup.....0fcda601dd289f83b20c250407eacfe7
- Full Text :
- https://doi.org/10.1016/j.jviromet.2006.02.004