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


OBJECTIVE
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.


CONCLUSIONS
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.
Schaefer
Stephan
S
Institute of Medical Virology, Justus Liebig University, Frankfurter Strasse 107, D-35392 Giessen, Germany.
Glebe
Dieter
D
Wend
Ulrike C
UC
Oyunbileg
J
J
Gerlich
Wolfram H
WH
D000818:Animals; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D016679:Genome, Viral; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics; D006801:Humans; D017918:Orthohepadnavirus / Q000145:classification* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D004279:DNA, Viral