Detection and analysis of diverse herpesviral species by consensus primer PCR.
A consensus primer PCR method which amplifies a region of herpesviral DNA-directed DNA polymerase (EC 2.7.7.7) and which uses degenerate primers in a nested format was developed. Primers were designed to target sequences coding for highly conserved amino acid motifs covering a region of approximately 800 bp. The assay was applied to 22 species of herpesviruses (8 human and 14 animal viruses), with PCR products obtained for 21 of 22 viruses. In the process, 14 previously unreported amino acid-coding sequences from herpesviral DNA polymerases were obtained, including regions of human herpesviruses 7 and 8. The 50 to 60 amino acid-coding sequences recovered in the present study were determined to be unique to each viral species studied, with very little sequence variation between strains of a single species when studied. Template dilution studies in the presence of human carrier DNA demonstrated that six human herpesviruses (herpesviruses 1, 2, 3, 4, 5, and 6B) could be detected at levels at or below 100 genome equivalents per 100 ng of carrier DNA. These data suggest that consensus primer PCR targeted to herpesviral DNA polymerase may prove to be useful in the detection and identification of known herpesviruses in clinical samples and the initial characterization of new herpesviral genomes.
VanDevanter
D R
DR
PathoGenesis Corporation, Seattle, Washington 98119, USA.
Warrener
P
P
Bennett
L
L
Schultz
E R
ER
Coulter
S
S
Garber
R L
RL
Rose
T M
TM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D005069:Evaluation Studies as Topic; D006564:Herpesviridae / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D017931:DNA Primers; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase