中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2013年
5期
461-466
,共6页
夏长胜%赵晓涛%孙媛媛%张正
夏長勝%趙曉濤%孫媛媛%張正
하장성%조효도%손원원%장정
巨细胞病毒%磷酸转移酶类(醇族体)%更昔洛韦%突变%抗药性,病毒%聚合酶链反应
巨細胞病毒%燐痠轉移酶類(醇族體)%更昔洛韋%突變%抗藥性,病毒%聚閤酶鏈反應
거세포병독%린산전이매류(순족체)%경석락위%돌변%항약성,병독%취합매련반응
Cytomegalovirus%Phosphotransferases (alcohol group acceptor)%Ganciclovir%Mutation%Drug resistance,viral%Polymerase chain reaction
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HCMV DNA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HCMV DNA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-限制性片段长度多态性分析方法(PCR-RFLP)检测UL97 M460V/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(ARMS RT-PCR)检测UL97 A594V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UL97 R494P、T502A、N558D及G561S突变.成功建立UL97 A594VARMS RT-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 ×102拷贝数/ml,检测筛选的临床标本发现2例患者为阳性,UL97 A594V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UL97 A594V耐药突变.
目的 瞭解造血榦細胞移植受者人巨細胞病毒(HCMV) UL97基因耐更昔洛韋(GCV)突變情況.方法 前瞻性收集2008至2010年在北京大學人民醫院血液病研究所行骨髓榦細胞移植術後血漿HCMV DNA暘性時間至少持續2週的患者43例,其中男29例,女14例,平均年齡21歲.利用實時熒光定量PCR檢測HCMV DNA的負荷,收集49份血漿標本檢測UL97耐GCV突變.應用改進的聚閤酶鏈反應-限製性片段長度多態性分析方法(PCR-RFLP)檢測UL97 M460V/I、H520Q、A591V、A594V、L595S/F及C603W共8種常見耐GCV突變；使用PCR直接測序法(PCR-DS)檢測UL97耐GCV突變；建立擴增阻滯突變繫統實時PCR方法(ARMS RT-PCR)檢測UL97 A594V耐藥突變.結果 PCR-RFLP分析方法未檢測齣8種已知的UL97耐GCV突變.PCR-DS方法未檢測到已知的UL97耐GCV突變,新髮現UL97 R494P、T502A、N558D及G561S突變.成功建立UL97 A594VARMS RT-PCR檢測方法,結閤覈痠提取試劑的使用,其檢測下限至少達7.5 ×102拷貝數/ml,檢測篩選的臨床標本髮現2例患者為暘性,UL97 A594V耐藥突變率為4.7％ (2/43).結論 構建的ARMSRT-PCR方法具有較高的敏感性,可用于臨床鑑測UL97 A594V耐藥突變.
목적 료해조혈간세포이식수자인거세포병독(HCMV) UL97기인내경석락위(GCV)돌변정황.방법 전첨성수집2008지2010년재북경대학인민의원혈액병연구소행골수간세포이식술후혈장HCMV DNA양성시간지소지속2주적환자43례,기중남29례,녀14례,평균년령21세.이용실시형광정량PCR검측HCMV DNA적부하,수집49빈혈장표본검측UL97내GCV돌변.응용개진적취합매련반응-한제성편단장도다태성분석방법(PCR-RFLP)검측UL97 M460V/I、H520Q、A591V、A594V、L595S/F급C603W공8충상견내GCV돌변；사용PCR직접측서법(PCR-DS)검측UL97내GCV돌변；건립확증조체돌변계통실시PCR방법(ARMS RT-PCR)검측UL97 A594V내약돌변.결과 PCR-RFLP분석방법미검측출8충이지적UL97내GCV돌변.PCR-DS방법미검측도이지적UL97내GCV돌변,신발현UL97 R494P、T502A、N558D급G561S돌변.성공건립UL97 A594VARMS RT-PCR검측방법,결합핵산제취시제적사용,기검측하한지소체7.5 ×102고패수/ml,검측사선적림상표본발현2례환자위양성,UL97 A594V내약돌변솔위4.7％ (2/43).결론 구건적ARMSRT-PCR방법구유교고적민감성,가용우림상감측UL97 A594V내약돌변.
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.
