Genotyping cytomegalovirus UL97 mutations by high-resolution melting analysis with unlabeled probe.
Human cytomegalovirus (CMV) is an opportunistic pathogen, and infections with this virus can be treated with ganciclovir (GCV). Most GCV-resistant clinical CMV isolates contain a mutation in the UL97 gene. Genotypic assays for diagnostic screening of GCV-resistant CMV have been developed. High-resolution melting analysis (HRMA) with unlabeled probe is considered a perfect tool for this purpose. In this study, we have developed an HRMA-based genotypic test for the detection of UL97 mutations. Wild type and M460V/I mutants of UL97 were constructed. HRMA with unlabeled probe was used as a genotyping method for the detection of M460V/I mutations. The melting peaks obtained directly from PCR products did not enable us to distinguish the wild type from M460 mutants. The sensitivity and accuracy of HRMA were dramatically improved by using unlabeled probe. HRMA with unlabeled probe successfully distinguished M460V from M460I and served well for the detection of M460V/I mutations in clinical samples. HRMA with unlabeled probe proves to be a sensitive and cost-effective genotyping method for the detection of M460 mutations.
Department of Clinical Laboratory, Peking University People's Hospital, No. 11, Xizhimen South Street, Peking, China.
Xiao-Tao
XT
Zhao
Dan-Qiu
DQ
Zhou
Shuai
S
Wu
Yue-Wen
YW
Chen
Yong
Y
Shao
Jie
J
Zhang
Chang-Sheng
CS
Xia
Ke-Peng
KP
Wang
Hong
H
Yang
Jun
J
Wan
Bo
B
Yu
Zheng
Z
Zhang
Wei
W
Zhang
D000998:Antiviral Agents / Q000494:pharmacology; D003587:Cytomegalovirus / Q000187:drug effects* / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D024882:Drug Resistance, Viral; D015774:Ganciclovir / Q000494:pharmacology; D005838:Genotype; D006801:Humans; D058889:Molecular Typing; D020125:Mutation, Missense; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D014773:Virology / Q000379:methods*
D000998:Antiviral Agents; D004279:DNA, Viral; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D015774:Ganciclovir