Identification of Human Cytomegalovirus pUL27 R233 point mutation using PCR-RFLP

Human Cytomegalovirus (HCMV) is one of the life-threatening agents in immunosuppressed patients and congenitally infected neonates in the world. Mutations in UL27 were suggested to confer low- to high-grade Maribavir (MBV) resistance. As pUL27 R233S variation may involve in either MBV-resistance, we aimed to establish a method for identifying R233 coding sequence mutation.
Eleven boiled-DNA extracts from 2000 congenitally CMV infected (cCMV) neonates urines were provided. Polymerase Chain Reaction (PCR) was performed to amplify R233 coding sequence. Restriction Fragment Length Polymorphism (RFLP), after selection of HhaI as a proper cutting enzyme at given site by NEBcutter server, was performed. PCR amplicons and digested samples were run on gel-electrophoresis for demonstration expected fragments.
Our result has proved that HhaI can cut UL27 containing wild type R233 coding sequence but not theR233 mutants. Among eleven clinical samples, one has shown R233 mutation, but other 10 samples had no variations by PCR-RFLP.
It seems that HhaI can be employed for molecular examination of HCMV UL27 R233 variations and this is the first report demonstrating that PCR-RFLP can be used to recognize CMV-pUL27 R233 mutation. Therefore, this work can open a new window for HCMV UL27 polymorphism analysis in the future.