Quantitative determination of human cytomegalovirus target sequences in peripheral blood leukocytes by nested polymerase chain reaction and temperature gradient gel electrophoresis

Abstract

A competitive nested PCR-temperature gradient gel electrophoresis protocol (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 gradient gel electrophoresis (TGGE). The number of HCMV target sequences 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 found to be five to 10 copies of the target sequence. Effects of sample preparation on quantitative HCMV PCR were minimized by the additional quantification of beta-globin target sequences and calculation of the ratio of HCMV copies/beta-globin copies. Serial peripheral blood leukocyte specimens of 17 renal allograft recipients positive in a qualitative nested HCMV PCR were tested using nPCR/TGGE. Thirty healthy blood donors served as negative controls. Positive results were obtained by nPCR/TGGE in nine renal allograft recipients but in none of the healthy blood donors. Five of five patients with an HCMV pp65 antigenaemia and positive for HCMV IgM were positive in nPCR/TGGE. The highest HCMV/beta-globin ratios (10,000 to 8000 copies HCMV/10(6) copies beta-globin) were found in transplant recipients experiencing acute clinically symptomatic HCMV infection. HCMV DNA levels in asymptomatic patients ranged from 900 to 200 copies HCMV/10(6) beta-globin.