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A sensitive direct sequencing assay based on nested PCR for the detection of HBV polymerase and surface glycoprotein mutations
- Source :
- Journal of Virological Methods. 159:53-57
- Publication Year :
- 2009
- Publisher :
- Elsevier BV, 2009.
-
Abstract
- Drug resistance is a crucial problem emerging frequently during treatment of hepatitis B, resulting in treatment failure and progression of liver damage. A direct sequencing method based on a nested PCR was established to detect mutations in samples with low viral load. Primers were designed to obtain an amplicon encompassing the A, B, C, D and E functional domains of HBV polymerase. Fifty-five samples were tested, containing HBV DNA ranging from 19 to 1700 IU/mL. Sixteen samples were also tested by the commercially available assay INNO-LiPA HBV DR v2. Sequencing was successful for all samples, and mutations were detected in 24/55 (43.6%). When used in parallel with DR v2, concordant results were found in 8/16 samples. In the eight discordant cases, four were resolved by sequencing and not by DR v2, and four had differences in the mutation patterns. Direct sequencing was able to show pol mutations not revealed by DR v2, such as rtV214A, rtQ215H/S, and rtM250V. Genotype and env variations were also established. This highly sensitive sequencing protocol, providing valuable sequencing data from samples with a low viral load, is suitable for detection of mutations at the very early signs of failure of treatment, thereby allowing to maximize the success of early treatment changes.
- Subjects :
- Hepatitis B virus
Gene Products, pol
Biology
medicine.disease_cause
Antiviral Agents
Polymerase Chain Reaction
Sensitivity and Specificity
law.invention
Open Reading Frames
Hepatitis B, Chronic
law
Virology
Drug Resistance, Viral
Genotype
medicine
Humans
Treatment Failure
Polymerase
Polymerase chain reaction
Mutation
Membrane Glycoproteins
Sequence Analysis, DNA
Viral Load
Amplicon
Molecular biology
biology.protein
Nested polymerase chain reaction
Viral load
Subjects
Details
- ISSN :
- 01660934
- Volume :
- 159
- Database :
- OpenAIRE
- Journal :
- Journal of Virological Methods
- Accession number :
- edsair.doi.dedup.....33ff9912f6f3a89695a1978f74b5a3c5
- Full Text :
- https://doi.org/10.1016/j.jviromet.2009.02.027