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J Clin Virol. 2003 May;27(1):30-7.

Universal primers for real-time amplification of DNA from all known Orthohepadnavirus species.

Author information

1
Institute of Medical Virology, Justus Liebig University, Frankfurter Strasse 107, D-35392 Giessen, Germany.

Abstract

BACKGROUND:

The family of Hepadnaviridae is made up of members infecting birds (genus Avihepadnavirus) or mammals (genus Orthohepadnavirus). Hepatitis B virus (HBV), the hepadnavirus infecting humans, can be divided into the seven genotypes A-G. By definition, genotypes differ by more than 8% at the nucleotide level. However, some genotypes differ by more than 14% from others.

OBJECTIVES:

The diversity of HBV genotypes necessitates great care in primer design to find primers suitable for routine diagnostic procedures that are highly conserved. Our aim was to find a target sequence on the HBV genome that is highly conserved among all known orthohepadnaviruses, to avoid false-negative polymerase chain reaction (PCR) results due to uncommon variants of HBV.

METHODS:

Using an alignment of 177 genomes of orthohepadnaviruses from GenBank, we selected a primer pair from a highly conserved region, corresponding to hydrophobic transmembrane domains of the major surface protein of HBV.

RESULTS:

The primer pair chosen was suitable to amplify genome sequences from HBV and to the genetically most distant woodchuck hepatitis virus in real-time PCR using the LightCycler, Roche. Moreover, the primers were suitable for accurate quantitation of both viral genomes over a range from 100 to 10(10) genomes/ml.

CONCLUSION:

The described primers are useful for reliable detection and accurate quantitation of all known hepadnaviral genomes and may be used for the search for unknown orthohepadnaviruses.

PMID:
12727526
[Indexed for MEDLINE]

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