人巨细胞病毒UL97基因耐更昔洛韦突变检测的研究

Detection of human cytomegalovirus UL97 gene mutations conferring ganciclovir resistance

目的了解造血干细胞移植受者人巨细胞病毒（HCMV）UL97基因耐更昔洛韦（GCV）突变情况。方法前瞻性收集2008至2010年在北京大学人民医院血液病研究所行骨髓干细胞移植术后血浆HCMVDNA阳性时间至少持续2周的患者43例，其中男29例，女14例，平均年龄21岁。利用实时荧光定量PCR检测HCMVDNA的负荷，收集49份血浆标本检测UL97耐GCV突变。应用改进的聚合酶链反应一限制性片段长度多态性分析方法（PCR—RFLP）检测UL97M460V／I、H520Q、A591V、A594V、L595S／F及C603W共8种常见耐GCV突变；使用PCR直接测序法（PCR—DS）检测UL97耐GCV突变；建立扩增阻滞突变系统实时PCR方法（ARMSRT—PCR）检测UL97A594V耐药突变。结果PCR—RFLP分析方法未检测出8种已知的UL97耐GCV突变。PCR—DS方法未检测到已知的uL97耐GCV突变，新发现UL97R494P、T502A、N558D及G561S突变。成功建立UL97A594VARMSRT—PCR检测方法，结合核酸提取试剂的使用，其检测下限至少达7．5×10^2拷贝数／ml，检测筛选的临床标本发现2例患者为阳性，UL97A594V耐药突变率为4．7％（2／43）。结论构建的ARMSRT—PCR方法具有较高的敏感性，可用于临床监测UL97A594V耐药突变。

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 7.5×10^2 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.