BACKGROUND: The development of infections with ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) remains a serious problem in recipients of stem cell or organ transplants. Nearly all GCV-resistant clinical isolates have mutations in the viral UL97 gene. The rapid detection of GCV-resistant HCMV infections is necessary and the relative proportions of wild-type and mutant strains are predictive for the efficiency of antiviral therapy. To date, genotypical resistance screening has been limited to restriction fragment length polymorphism (RFLP) and sequencing analyses. Here, we present a comprehensive real-time PCR approach for the detection of most frequent mutations in the UL97 gene associated with GCV resistance. METHODS: The laboratory strains AD169 and Towne, different wild-type isolates and plasmids constructed by site-directed mutagenesis and overlap extension with specific point-mutations in the UL97 gene were analysed by LightCycler PCR and compared with UL97 RFLP and sequencing analyses. RESULTS: A new and comprehensive set of LightCycler PCRs was created using specific hybridization probes with melting-point analysis for the relevant codons 594, 595, 603 and 607. Different wild-type isolates and plasmids containing specific UL97 mutations conferring GCV resistance were investigated in the real-time PCR assay. Total processing time was 80 min per assay, whereas combinations of RFLP and sequencing needed at least 3-4 days. Proportions of co-existing wild-type and mutant strains in mixed viral populations can be obtained. CONCLUSIONS: We established a rapid real-time PCR approach for the detection of most frequent HCMV UL97 mutations associated with GCV resistance. Moreover, the method allows semiquantitative differentiation of the proportions of co-existing wild-type and mutant strains. This approach represents a new alternative for laborious RFLP analysis.
BACKGROUND: The development of infections with ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) remains a serious problem in recipients of stem cell or organ transplants. Nearly all GCV-resistant clinical isolates have mutations in the viral UL97 gene. The rapid detection of GCV-resistant HCMVinfections is necessary and the relative proportions of wild-type and mutant strains are predictive for the efficiency of antiviral therapy. To date, genotypical resistance screening has been limited to restriction fragment length polymorphism (RFLP) and sequencing analyses. Here, we present a comprehensive real-time PCR approach for the detection of most frequent mutations in the UL97 gene associated with GCV resistance. METHODS: The laboratory strains AD169 and Towne, different wild-type isolates and plasmids constructed by site-directed mutagenesis and overlap extension with specific point-mutations in the UL97 gene were analysed by LightCycler PCR and compared with UL97 RFLP and sequencing analyses. RESULTS: A new and comprehensive set of LightCycler PCRs was created using specific hybridization probes with melting-point analysis for the relevant codons 594, 595, 603 and 607. Different wild-type isolates and plasmids containing specific UL97 mutations conferring GCV resistance were investigated in the real-time PCR assay. Total processing time was 80 min per assay, whereas combinations of RFLP and sequencing needed at least 3-4 days. Proportions of co-existing wild-type and mutant strains in mixed viral populations can be obtained. CONCLUSIONS: We established a rapid real-time PCR approach for the detection of most frequent HCMVUL97 mutations associated with GCV resistance. Moreover, the method allows semiquantitative differentiation of the proportions of co-existing wild-type and mutant strains. This approach represents a new alternative for laborious RFLP analysis.