Value of a ligase chain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirus.
Human cytomegalovirus (HCMV) isolates resistant to ganciclovir were found in patients undergoing therapy. Therefore, we have developed a new specific and sensitive method--a ligase chain reaction (LCR) assay--for detection of frequently encountered 594 mutated codon in ganciclovir (GCV) resistant virus. Previous studies characterized an alanine to valine change on codon 594 in resistant strains. A novel substitution in 594, alanine to glycine, is described which is also capable of conferring ganciclovir resistance. LCR products were analyzed on polyacrylamide gel- and the mutant was detected using a non radioactive method. The LCR product detection was then adapted to a microtitre plate format with a colorimetric detection. This method allowed the distinction of mutated GCV-resistant strains from sensitive strains with a high sensitivity, and the detection of a low percentage of mutated DNA in virus load. This assay could be useful in following the evolution of mutated DNA compared to viral infection.
Bourgeois
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Laboratoire de Virologie, Faculté de Médecine, Dijon, France.
Sixt
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Bour
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Pothier
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D000409:Alanine / Q000235:genetics; D003587:Cytomegalovirus / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D004352:Drug Resistance, Microbial; D004591:Electrophoresis, Polyacrylamide Gel; D015774:Ganciclovir / Q000494:pharmacology*; D005814:Genes, Viral; D005998:Glycine / Q000235:genetics; D006801:Humans; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D015678:Viral Structural Proteins / Q000235:genetics*
D015678:Viral Structural Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D000409:Alanine; D015774:Ganciclovir; D005998:Glycine