Detection of human cytomegalovirus UL97 gene mutations conferring ganciclovir resistance / 中华检验医学杂志

ABSTRACT

Objective To explore human cytomegalovirus UL97 mutations related to ganciclovir resistance in hematopoietic stem cell transplant (HSCT) recipients.Methods A total of 43 patients,including 24 males and 19 females,with an average age of 21 years old,who had HCMV DNAemia for more than two weeks after HSCT between 2008 and 2010 in Peking University People's Hospital,were included in this prospective study.UL97 GCV resistance mutations were investigated in 49 plasma specimens collected from those patients.GCV resistance mutations such as UL97 M460V/I,H520Q,A591V,A594V,L595S/F,and C603W,were analyzed by modified PCR-RFLP methods.UL97 mutations related to GCV resistance were assayed by the method of PCR-direct sequencing (PCR-DS).An amplified refractory mutation system real-time PCR (ARMS RT-PCR) was developed for the detection of UL97 A594V mutation.Results Eight known UL97 ganciclovir resistance mutations were not detected by PCR-RFLP and PCR-DS.Four new UL97 mutations like UL97 R494P,T502A,N558D,and G561S,were detected by PCR-DS.The ARMS RT-PCR for detecting of UL97 A594V was established successfully.The lower limit of detection of the method was at least 7.5 × 102 copies/ml combined with the use of nucleic acid extraction reagent.UL97 A594V resistance mutation was identified by the method of ARMS RT-PCR in two HSCT recipients.The rate of UL97 A594V mutation was 4.7％ (2/43) in HSCT recipients.Conclusion The ARMS RT-PCR assay represented a sensitive method for the identification of UL97 A594V mutation.