QUANTITATIVE-DETERMINATION OF HUMAN CYTOMEGALOVIRUS TARGET SEQUENCES IN PERIPHERAL-BLOOD LEUKOCYTES BY NESTED POLYMERASE CHAIN-REACTION ANDTEMPERATURE-GRADIENT GEL-ELECTROPHORESIS

A competitive nested PCR-temperature gradient gel electrophoresis prot ocol (nPCR/TGGE) has been established for the quantification of human cytomegalovirus (HCMV) target sequences. The measurement was achieved by co-amplification of a defined copy number of an internal standard ( st) and separation of st and wild-type (wt) amplimers by temperature g radient gel electrophoresis (TGGE). The number of HCMV target sequence s could be precisely determined within wt/st ratios of 0.1 to 10. With 50 copies of the st sequence the detection limit of nPCR/TGGE was fou nd to be five to 10 copies of the target sequence. Effects of sample p reparation on quantitative HCMV PCR were minimized by the additional q uantification of beta-globin target sequences and calculation of the r atio of HCMV copies/beta-globin copies. Serial peripheral blood leukoc yte specimens of 17 renal allograft recipients positive in a qualitati ve nested HCMV PCR were tested using nPCR/TGGE. Thirty healthy blood d onors served as negative controls. Positive results were obtained by n PCR/TGGE in nine renal allograft recipients but in none of the healthy blood donors. Five of five patients with an HCMV pp65 antigenaemia an d positive for HCMV IgM were positive in nPCR/TGGE. The highest HCMV/b eta-globin ratios (10000 to 8000 copies HCMV 10(6) copies beta-globin) were found in transplant recipients experiencing acute clinically sym ptomatic HCMV infection. HCMV DNA levels in asymptomatic patients rang ed from 900 to 200 copies HCMV/10(6) beta-globin.