Quantitation of human cytomegalovirus glycoprotein H gene in cells using competitive PCR and a rapid fluorescence-based detection system.
A technique is described for quantitation of the human cytomegalovirus (HCMV) glycoprotein H (gH) gene in cells using a quantitative-competitive polymerase chain reaction (QC-PCR). Two recombinant DNA molecules, differing in size due to a 92-bp deletion within the HCMV gH sequence, were used in co-amplification studies to construct a standard curve from which the copy number of the gH gene present in clinical samples could be interpolated. The use of primers labeled with a fluorescent dye allowed direct detection of the amplified products by measuring the amount of fluorescence emitted by each specific PCR fragment with an automated DNA sequencer coupled to a software program. This system was validated subsequently using bronchoalveolar lavage cells obtained from immunocompromised patients and found to be highly sensitive and reproducible over a range of 5-50,000 HCMV gH copies. This rapid procedure could easily be applied to study the pathogenesis of HCMV infection, identify the patients at high risk of developing HCMV disease, and monitor the effects of antiviral therapy at the molecular level.
Boivin
G
G
Department of Medicine, University of Minnesota Medical School, Minneapolis, USA.
Olson
C A
CA
Quirk
M R
MR
St-Cyr
S M
SM
Jordan
M C
MC
D001483:Base Sequence; D015139:Blotting, Southern; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics* / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000175:diagnosis / Q000821:virology; D017931:DNA Primers; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis; D005453:Fluorescence; D005814:Genes, Viral / Q000235:genetics*; D006801:Humans; D016867:Immunocompromised Host; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017384:Sequence Deletion / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*
D017931:DNA Primers; D004274:DNA, Recombinant; D004279:DNA, Viral; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins; C071440:glycoprotein H, Cytomegalovirus; C499358:glycoprotein H, Human cytomegalovirus