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

• Published in: Journal of clinical virology Vol. 27; no. 1; pp. 30 - 37
• Main Authors: Schaefer, Stephan, Glebe, Dieter, Wend, Ulrike C., Oyunbileg, J., Gerlich, Wolfram H.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.05.2003
• Subjects: Animals; DNA Primers; DNA, Viral - analysis; DNA, Viral - genetics; Genome, Viral; Hepatitis B virus; Hepatitis B virus - genetics; Hepatitis B Virus, Woodchuck - genetics; Humans; LightCycler; Orthohepadnavirus; Orthohepadnavirus - classification; Orthohepadnavirus - genetics; Polymerase Chain Reaction - methods; Real-time PCR; Woodchuck hepatitis virus; Orthohepadnavirus; Hepatitis B virus; Real-time PCR; LightCycler; Woodchuck hepatitis virus
• ISSN: 1386-6532; 1873-5967
• DOI: 10.1016/S1386-6532(02)00108-7

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.