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 wildtype (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 l0 copies of the target sequence. Effects of sample preparation on quantitative HCMV PCR were minimized by the additional quantification offl-globin target sequences and calculation of the ratio of HCMV copies/fl-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/fl-globin ratios (10000 to 8000 copies HCMV/ 106 copies fl-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 ~ fl-globin.

Longitudinal analysis of cytomegalovirus load in renal transplant recipients using a quantitative polymerase chain reaction: correlation with disease

“…However, in the current study, statistically significant differences in viral load between the asymptomatic and symptomatic patients were observed. Other studies have used similar and alternative methods for quantification of HCMV including semi-quantitative PCR (Cagle et al, 1992), quantitative dot-blot hybridization (Saltzman et al, 1992), quantitative antigenemia (Landry & Ferguson, 1993) and competitive nested PCR (Gerdes et al, 1993;Schafer et al, 1993). In the study of Saltzman et al (1992), AIDS patients and solid organ recipients with visceral HCMV disease had higher levels of genomes in the blood than similar patients without visceral involvement.…”

“…In the study of Saltzman et al (1992), AIDS patients and solid organ recipients with visceral HCMV disease had higher levels of genomes in the blood than similar patients without visceral involvement. In the study by Schafer et al (1993), blood from renal transplant patients was analysed and in three patients with symptomatic infection from a total study group of 17, HCMV load in the blood was approximately 1 log10 higher than in patients who remained asymptomatic. Taken together, the results described here coupled with those from other workers (vide supra) clearly demonstrate that HCMV load is an important factor in HCMV pathogenesis and disease.…”

“…The latter method is proving to be the most accurate method for quantification and has been applied to many viral systems including HCMV Schafer et al, 1993). The HCMV method previously reported by our laboratory utilizes a control sequence within each PCR that is identical to the target sequence except for the modification of two base pairs to yield a restriction site for HpaI.…”

Transfusion-Associated Infections with Cytomegalovirus and Other Human Herpesviruses

“…In contrast, serologic assays are largely unsuitable for the diagnosis of exogenous reinfection with HCMV or endogenous reactivation. In these cases, laboratory diagnosis has to rely on the quantitative determination of viral DNA levels, on the detection of viral transcripts or antigens, and/or on virus isolation [11,12]. The reliable discrimination of exogenous HCMV reinfection from endogenous reactivation may be achieved by methods of molecular epidemiology, e.g., different HCMV genotypes can be discriminated by restriction fragment length polymorphism analysis, separation of PCR products by denaturing gradient gel electrophoresis, or sequencing of genome fragments.…”

Comparison of three PCR techniques for detecting cytomegalovirus (CMV) DNA in serum, detection of early antigen fluorescent foci and culture for the diagnosis of CMV infection

“…To differentiate between sample and control targets, previous workers have created restriction sites by in-vitro mutagenesis [22], introduced single-based permutations and separated products by temperature gradient gel electrophoresis [23], introduced heterologous DNA and differentiated between products by hybridisation [24,25] or generated products of different sizes, easily separated by gel electrophoresis [ 1 1, 25, 261.…”