Quantitative PCR for human herpesviruses 6 and 7.
A quantitative PCR assay for the detection of human herpesvirus 6 (HHV-6) (variants A and B) and HHV-7 DNAs in clinical samples was developed. The assay uses a nonhomologous internal standard (IS) for each virus that is coamplified with the wild-type target sequence in the same vial and with the same pair of primers. This method allows for a correction of the variability of efficiency of the PCR technique. A standard curve is constructed for each experiment by coamplification of known quantities of the cloned HHV-6 or HHV-7 target templates with the respective IS. Absolute quantitation of the test samples is then achieved by determining the viral target/IS ratio of the hybridization signals of the amplification products and plotting this value against the standard curve. Using this assay, we quantitated the amount of HHV-6 or HHV-7 DNA in infected cell cultures and demonstrated an inhibitory effect of phosphonoformic acid on the replication of HHV-6 and HHV-7 in vitro. As the first clinical application of this procedure, we performed preliminary measurements of the loads of HHV-6 and HHV-7 in lymph nodes from patients with Hodgkin's disease and AIDS. Application of this quantitative PCR method should be helpful for elucidating the pathogenic roles of HHV-6 and HHV-7.
Secchiero
P
P
Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesda, Maryland 20892-4255, USA.
Zella
D
D
Crowley
R W
RW
Gallo
R C
RC
Lusso
P
P
D000163:Acquired Immunodeficiency Syndrome / Q000150:complications / Q000821:virology; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005069:Evaluation Studies as Topic; D006566:Herpesviridae Infections / Q000150:complications / Q000175:diagnosis; D015654:Herpesvirus 6, Human / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D016199:Herpesvirus 7, Human / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D006689:Hodgkin Disease / Q000150:complications / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards
D017931:DNA Primers; D004279:DNA, Viral