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-competi tive polymerase chain reaction (QC-PCR). Two recombinant DNA molecules , differing in size due to a 92-bp deletion within the HCMV gH sequenc e, were used in coamplification 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 d ye 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 fr om immunocompromised patients and found to be highly sensitive and rep roducible over a range of 5-50 000 HCMV gH copies. This rapid procedur e could easily be applied to study the pathogenesis of HCMV infection, identify the patients at high risk of developing HCMV disease, and mo nitor the effects of antiviral therapy at the molecular level.