Rapid detection of codon 460 mutations in the UL97 gene of ganciclovir-resistant cytomegalovirus clinical isolates by real-time PCR using molecular beacons.
A rapid real-time polymerase chain reaction (PCR) assay using molecular beacons has been developed for the simultaneous detection of wild-type and mutant strains of cytomegaloviruses (CMV) with respect to codon 460 of the UL97 gene has been developed. The molecular beacons were designed to complement the wild-type codon 460 or the mutant sequence arising from a single base-pair difference (point mutation). Discrimination between wild-type and mutant templates was demonstrated as the beacons did not generate fluorescence with their respective mismatch targets but only with those that they were designed to perfectly anneal with. Samples that harbor mixed populations of CMV could also be readily recognized. Applied to a small number of clinical samples, the retrospective screening by this assay are in general concordance with that obtained by PCR-RFLP. Using molecular beacons strategy, codon 460 mutation was detected in ten out o the total number of 40 samples, whereas the latter method identified nine samples as containing the mutation. The discrepant result arose from the genotyping of one clinical sample as mixed (containing both wild-type and mutant CMV strains) by molecular beacons but as wild-type by PCR-RFLP, suggesting that this real-time strategy is possibly more sensitive for mutation analysis.
Yeo
Adrian C
AC
Department of Pediatrics, National University of Singapore, Singapore; School of Chemical and Life Sciences, Singapore Polytechnic, 500 Dover Road, 138651 Singapore. mahler@sp.edu.sg
Chan
Kwai Peng
KP
Kumarasinghe
Gamini
G
Yap
Hui Kim
HK
D019943:Amino Acid Substitution / Q000235:genetics; D000998:Antiviral Agents / Q000494:pharmacology; D003062:Codon; D003587:Cytomegalovirus / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics*; D015774:Ganciclovir / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008715:Methionine / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D014633:Valine / Q000235:genetics
D000998:Antiviral Agents; D003062:Codon; D004279:DNA, Viral; D008715:Methionine; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D014633:Valine; D015774:Ganciclovir