Development of SYBR Green I-based real-time PCR assay for detection of drug resistance mutations in cytomegalovirus

Ganciclovir (GCV) is an antiviral drug that is used to treat cytomegalovirus (CMV) infection. However, long-term monotherapy does not commonly result in complete suppression of viral replication and is associated with the emergence of resistant mutants. In this study, a method for detecting CMV resistance mutations was carried out by real-time amplification refractory mutation system PCR (real-time ARMS PCR) using SYBR Green I fluorescent dye. Three recombinant plasmids were constructed by overlapping extension PCR to be used as standard mutation or wild-type models. Four pairs of primers were used to amplify the approximately 150 bp of the UL97 gene spanning codon 460, where mutations associated with resistance to GCV invariably occur. As little as 20% mutants DNA in 107 copies/ml mixture DNA were detected. Though this approach was not more sensitive than PCR-restriction fragment length polymorphism (RFLP) for the detection of the presence of mixtures, it was a high-throughput and automation method, and the specific mutation type can be deduced by the real-time ARMS PCR data. Overall, this study has demonstrated an approach that could be a sensitive and rapid method for the detection of GCV resistance-associated mutation in CMV.