PCR with quenching probes enables the rapid detection and identification of ganciclovir-resistance-causing U69 gene mutations in human herpesvirus 6

A single-nucleotide polymorphism detection assay using PCR with quenching probes (QP-PCR) was developed for the rapid detection of antiviral drug-resistance mutations of human herpesvirus 6 (HHV-6). The mutations examined were in the HHV-6 U69 gene, and were single-base mutations in sequences known to be associated with ganciclovir (GCV) resistance in HCMV. We previously confirmed that they conferred GCV resistance to recombinant baculoviruses (Nakano et al., J. Virol. Methods 161:223–230, 2009). Six characterized mutations, including a previously reported one that encodes a GCV-sensitive kinase-activity mutant (Isegawa et al., J. Clin. Virol. 44:15–19, 2009), were used. The six mutations were separated into three groups based on their location in the U69 protein, and detected by the hybridization of three probes. We developed and validated a set of assays for these mutations using PCR followed by differential melting of a fluorescently labeled oligo probe, on a Roche Light Cycler platform. Nucleobase quenching was used to detect the hybridized probe. The optimized assay could distinguish the different mutants, and easily detected mutants representing 30% of the DNA in a mixed sample. This QP-PCR assay permitted the rapid (1.5 h), objective, and reproducible detection of drug-resistant mutations of HHV-6.