Background: Human cytomegalovirus (CMV) is an opportunistic pathogen that can be treated with ganciclovir. Mutations in the UL97 gene of CMV render the virus ganciclovir resistance. These include H520Q and C603W mutations, against which we developed a novel genotyping assay for their identification.
Methods: PCR reactions were performed to amplify fragments of the UL97 gene containing H520Q or C603W mutations. High resolution melting analysis (HRMA) coupled with unlabeled DNA probes was employed to identify the shift in melting temperature of the probe-template complex, which reflexes the presence of point mutations.
Results: Melting point analysis performed on the dimeric DNA of PCR products of UL97 gene could not identify mutations in the gene. When coupled to unlabeled probes, point mutations in UL97 can be identified by analyzing the melting curve of probe-template complex. When WT and mutant UL97 DNAs were mixed together to mimic heterogeneous viral population in clinical samples, the genotyping assay is sensitive enough to detect H520Q and C603W mutants that constitute 10% of total DNA input.
Conclusion: Probe-based HRMA is effective in detecting H520Q and C603W mutations in the UL97 gene of CMV.
Keywords: C603W; H520Q; UL97; cytomegalovirus; high resolution melting analysis.
© 2015 Wiley Periodicals, Inc.