人巨细胞病毒对更昔洛韦耐受性的基因型鉴别方法的建立和应用

目的建立实验室检测人巨细胞病毒（HCMV）更昔洛韦（GCV）耐药的基因型分析的常规方法。方法采用巢式聚合酶链反应（PCR）直接扩增33例HCMV感染患者的外周血白细胞及前期实验同步获得的33例临床分离毒株的HCMVUL97基因片段，对扩增产物进行测序，与HCMV标准毒株AD169序列比对，参照国外报道的耐药相关的热点突变，判定各临床分离毒株的耐药基因型。结果除有1株为HCMVUL97呈M460V突变型外，余32株同标准株AD169的序列一致。同一患者的分离毒株与外周血白细胞中测得HCMVUL97基因序列完全一致。结论建立的实验室检测HCMV耐药的基因型分析完全可以替代传统的耐药表型分析，前者无需分离活病毒，不仅需时短，而且相对安全，其技术要点更适宜临床推广应用。

The establishment and application of the method for identifying ganciclovir-resistant genotypes of Human cytomegalovirus

Objective To establish a routine method for analyzing ganciclovir（GCV）-resistant genotypes of Human cytomegalovirus （HCMV）. Methods DNA extracted from peripheral leukocytes of 33 patients with HCMV infection and 33 clinical isolated strains were used to amplify UL97 gene by nested polymerase chain reaction（ PCR）, then they were sequenced. After making a contrast with AD169, the significant mutants were found according to verifying drug-resistant sites. Results After amplifying and sequencing, only one drug-resistant mutant （M460V） was found. The rest of them had sequences identical to AD169. UL97 gene from different samples of one patient was invariable. Conclusions Genotypic assay for drug-resistant detection can completely replace the phenotypic method for clinical routine use due to its advantages, such as rapidity, simplicity, precision, which are more suitable for clinical application.