骨髓移植患者血及尿中巨细胞病毒感染核酸标志物的检测

目的 为骨髓移植患者寻找较好的诊断和治疗巨细胞病毒（ＣＭＶ）感染的分子生物学依据。方法 用聚合链反应（ＰＣＲ）方法测尿中ＣＭＶ－ＤＮＡ,逆转录（ＲＴ）－ＰＣＲ法测血中ＣＭＶ－即刻早期抗原（ＩＥ）ｍＲＮＡ。结果 在被测的２７例患者１０３份标本中,发现１０例ＣＭＶ感染者的尿ＣＭＶ－ＤＮＡ及血ＩＥ ｍＲＮＡ几乎同时阳转。在连续使用抗病毒药甘昔洛瓦（ＤＨＰＧ）治疗的６例患者中,ＩＥ ｍＲＮＡ约一周左右阴转     

巨细胞病毒即刻早期基因mRNA检测方法的建立

目的建立快速简便的逆转录聚合酶链反应（ＲＴＰＣＲ）方法检测人巨细胞病毒（ＨＣＭＶ）即刻早期（ＩＥ）基因ｍＲＮＡ。方法设计合成跨越ＨＣＭＶＩＥ基因内含子区的一对引物，分别用ＲＴＰＣＲ和ＰＣＲ技术扩增ＨＣＭＶｍＲＮＡ和ＤＮＡ，用Ｓｏｕｔｈｅｒｎ杂交鉴定阳性产物。结果ＨＣＭＶＩＥ基因ｍＲＮＡ表达在感染后６小时即可出现，并持续到感染后至少９６小时，ＲＴＰＣＲ检测的最低敏感性为１００ｆｇ总ＲＮＡ，其他病毒和细胞ＲＮＡ不能被扩增。结论ＲＴＰＣＲ技术检测ＨＣＭＶｍＲＮＡ敏感、特异，有望应用于临床作为ＨＣＭＶ活动性感染的早期诊断方法     

套式PCR快速诊断新生儿巨细胞病毒感染

套式ＰＣＲ用于检测新生儿及随访ＣＩＤ患儿白细胞及尿中ＨＣＭＶＤＮＡ。两对引物序列取自ＨＣＭＶＩＥＡ基因区，第一对引物扩增７２１ｂｐ片段，第二对引物扩增１６７ｂｐ片段，后者穴居于前者之中，结果３３例可疑新生患儿检出ＣＭＶ感染２１例，７例随访患儿中４例尿或血中仍有ＣＭＶＤＮＡ存在。ＰＣＲ与病毒分离相比，既快速又敏感，尿标本用于ＰＣＲ检测ＣＭＶＤＮＡ较之血本具有检出率高，标本采取及处理均简便的优点。     

巨细胞病毒即刻早期及晚期基因片段的检测

自巨细胞病毒（ＨＣＭＶ）ＡＤ１６９株感染细胞中提取的ＤＮＡ，应用聚合酶链反应（ＰＣＲ）技术，在自行设计及合成的两对特异性引物引导下，成功地扩增出口ＣＭＶ即刻早期（ＩＥ）及晚期（Ｌ）基因片段，摸索了最佳Ｔａｑ酶、Ｍｇ￣（２＋）浓度及退火温度的反应条件，并建立了同一体系中同时检测ＨＣＭＶＩＥ及Ｌ基因片段的方法。初步应用该方法检测了１２例肾移植患者尿标本及１０例动脉粥样硬化标本中ＨＣＭＶＩＥ及Ｌ基因，结果ＩＥ阳性率分别为８％及２０％，Ｌ基因阳性率为３３％及５０％，未见有假阳性及假阴性结果的出现，表明该方法具有较好的特异性及灵敏度，可广泛地应用于临床ＨＣＭＶ的诊断。     

巨细胞病毒对人骨髓粒-巨噬系祖细胞生长的影响

目的探讨人巨细胞病毒（ＨＣＭＶ）对人骨髓粒巨噬系祖细胞集落（ＣＦＵＧＭ）是否有直接抑制作用。方法采用造血祖细胞体外培养技术,研究病毒株ＨＣＭＶＡＤ１６９对ＣＦＵＧＭ生长的影响,用原位聚合酶链反应（ＩＳＰＣＲ）技术检测ＣＦＵＧＭ的细胞内ＨＣＭＶＡＤ１６９ＤＮＡ。结果高浓度ＨＣＭＶＡＤ１６９（２×１０６ｐｆｕ／ｍｌ和２×１０５ｐｆｕ／ｍｌ）在体外能明显抑制ＣＦＵＧＭ的生长（Ｐ＜０．０１）,而低浓度（２×１０４ｐｆｕ／ｍｌ）ＨＣＭＶＡＤ１６９则无此作用,ＨＣＭＶＡＤ１６９对骨髓ＣＦＵＧＭ的抑制作用因其浓度的不同而有差异;经ＩＳＰＣＲ检测发现在ＣＦＵＧＭ的细胞核中存在ＨＣＭＶＡＤ１６９ＤＮＡ。结论ＣＦＵＧＭ是ＨＣＭＶＡＤ１６９的宿主细胞之一,ＨＣＭＶＡＤ１６９对骨髓ＣＦＵＧＭ生长的抑制作用与其对ＣＦＵＧＭ的直接感染有关。     

立即早期循环mRNA检测器官移植后患者的巨细胞病毒感染

立即早期循环ｍＲＮＡ检测器官移植后患者的巨细胞病毒感染（｜｜ＲａｎｄｈａｗａＰＳ，ｅｔａｌ．ＪＩｎｆＤｉｓ，１９９４；１７０：１２６４）ＰＣＲ扩增巨细胞病毒（ＣＭＶ）ＤＮＡ已被证实为诊断ＣＭＶ感染的敏感方法。但极敏感的ＰＣＲ法可在一些无临床症状者中检...     

套式聚合酶链反应检测巨细胞病毒的初步研究

目的建立套式聚合酶链反应（ＰＣＲ）加限制性酶切分析方法检测人巨细胞病毒（ＨＣＭＶ）。方法在ＨＣＭＶ直接早期蛋白ＥｃｏＲＩＪＤＮＡ片段内，自行设计两对引物，建立套式ＰＣＲ检测ＨＣＭＶＤＮＡ，同时结合病毒分离检测临床标本。结果２３例新生儿肝炎综合征患儿中，１０例病毒分离及套式ＰＣＲ均阳性；１例病毒分离阴性，但套式ＰＣＲ阳性。对５８例妊娠早期孕妇血标本进行检测，套式ＰＣＲ阳性率为９％，病毒分离阳性率则为７％。结论套式ＰＣＲ加限制性酶切分析是一种临床检测ＨＣＭＶ的快速有效手段，值得临床推广。     

应用PCR和ELISA法检测新生儿不同标本中的巨细胞病毒

为了解新生儿感染巨细胞病毒检测方法的可靠性，本文用ＥＬＩＳＡ法筛１８１８例孕妇，查出ＣＭＶ－ＩｇＭ阳性４６例，并以５０例ＣＭＶ－ＩｇＭ阴性作为对照组，在其分娩时用ＰＣＲ检测脐血、新生儿血、新生儿尿中ＣＭＶ－ＤＮＡ，用ＥＬＩＳＡ法测血清ＤＣＭＶ－ＩｇＭ。结果显示对照组两种方法均为阴性，实验组ＥＬＩＳＡ法阳性率５％，ＰＣＲ法阳性率２７％有显著差异。两种方法比较ＰＣＲ法具有高度特异性和敏感性。并提出先天     

人类第6型疱疹病毒感染与特发性血小板减少性紫癜

探讨人类第６型疱疹病毒（ＨＨＶ－６）感染与特发性血小板减少性紫癜（ＩＴＰ）的关系。方法采用聚合栈 反应（ＰＣＲ）方法检测１０５例ＩＴＰ患者骨髓单个核细胞（ＢＭＭＮＣ）ＨＨＶ－６ＤＮＡ及部分患者微小病毒Ｂ１９、巨细胞病毒（ＨＣＭＶ）ＤＮＡ序列;用竞争性ＥＬＩＳＡ方法检测６６例ＩＴＰ患者血小板相关抗体（ＰＡＩｇ）,并采用间接免疫荧光法动态观察１９例ＩＴＰ患者抗ＨＨＶ－６血清抗体滴度变化。结果①ＩＴＰ患     

应用PCR检测258例乙肝患者血清HBV-DNA结果报告

我院自１９９７年１月至１９９８年５月,应用聚合酶链反应（ＰＣＲ）技术检测乙型肝炎２５８例患者血清中ＨＢＶ－ＤＮＡ,并同时检测乙肝病毒标志物（ＨＢＶＭ）,现报告如下。材料和方法２５８例患者均为住院病人,按１９９０年上海全国病毒性肝炎会议制定的病毒性肝炎防治方案进行诊断,急性肝炎（ＡＨ）６５例;慢性肝炎（ＣＨ）１４５例,其中轻度７０例、中度４６例、重度２９例;肝炎肝硬化（ＨＬＣ）４８例。全部病例皆采用酶联免疫（ＥＬＩＳＡ）法检测ＨＢＶＭ,并排除甲、丙、Ｅ、Ｇ肝炎。ＰＣＲ和ＥＬＩＳＡ试剂盒均由河南省洛…     

Diagnostic value of nucleic-acid-sequence- based amplification for the detection of cytomegalovirus infection in renal and liver transplant recipients

To evaluate the diagnostic value of nucleic-acid-sequence-based amplification (NASBA) for the detection of cytomegalovirus (CMV) infection in transplant recipients, we compared immediate early 1 (IE1) and late pp67 mRNA detection by NASBA with the antigenemia assay, PCR and viral culture in 72 renal transplant (RTx) recipients and with antigenemia and serology in 25 liver transplant (LTx) recipients. Antigenemia, viral culture and pp67 NASBA were almost equivalent for the detection of CMV in RTx recipients. In LTx recipients, antigenemia detected more positive samples and more positive recipients compared to pp67 NASBA. In RTx recipients, PCR detected more positive samples and positive recipients compared to pp67 NASBA, antigenemia and viral culture. Also the first day of detection was slightly earlier for PCR. However, IE1 NASBA was the most sensitive test and detected 96% of all positive samples and positive transplant recipients. In addition, IE1 NASBA preceded PCR and all other positive results. This makes IE1 NASBA a very attractive screening test for the early detection of CMV infection.     

The detection of cytomegalovirus DNA by the polymerase chain reaction in different biological materials in patients with organ allografts

Ninety-eight patients with organ allografts were examined using the polymerase chain reaction (PCR). Cytomegalovirus (CMV) DNA was not found in the blood of any of 67 blood donors. At least 85% patients with CMV DNA in the plasma developed CMV disease. CMV DNA was detected in leukocytes of 22 (40.7%) out of 54 patients with negative results of analysis for CMV DNA in the plasma; 16 (29.6%) of these 22 presented with clinical symptoms of CMV infection by the moment of investigation. Results of PCR analysis of blood, urine, and saliva specimens of 30 patients examined did not always coincide. DNA isolated from the urine can contain components inhibiting DNA polymerase. Blood leukocytes or whole blood are the most suitable for the diagnosis of active and symptomatic CMV-infection.     

巨细胞病毒对多发性骨髓瘤细胞系KM3细胞的感染及其对IL-6 mRNA表达的影响

目的　探讨人巨细胞病毒 (CMV)对多发性骨髓瘤细胞系KM3细胞的感染及其对细胞IL 6mRNA表达的影响。方法　以 10 0 ,10 ,1半数组织培养感染量 (TCID50 )滴度的CMV与KM3细胞共培养 ,RT PCR法检测细胞CMV即刻早期抗原基因 (IE)、甘油醛 3 磷酸脱氢酶基因 (GAPDH)及IL 6mRNA的表达 ,流式细胞术检测细胞CMVpp6 5抗原的表达 ,透射电镜检测细胞内CMV病毒颗粒。结果　CMV感染的KM3细胞可明显地表达IEmRNA ,同时该细胞IL 6mRNA水平明显升高 ;10 0 ,10TCID50滴度的CMV感染的KM3细胞CMVpp6 5抗原表达率分别为 (5 .5 8± 1.5 5 ) %、(3.75± 0 .85 ) % ,与对照组的 (1.5 8± 0 .33) %相比 ,差异有显著性 (P <0 .0 5 ) ;透射电镜下 ,10 0TCID50 滴度的CMV感染的KM3细胞内及胞膜表面可见到CMV病毒颗粒。结论　CMV可感染多发性骨髓瘤细胞系KM3细胞 ,并在其中活化复制 ;该病毒可提高KM3细胞IL 6mRNA的表达     

Early detection of plasma cytomegalovirus DNA by real-time PCR after allogeneic hematopoietic stem cell transplantation

Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Therefore, preemptive ganciclovir therapy based on early detection of CMV reactivation is widely used to prevent CMV disease. Real-time polymerase chain reaction (PCR) has been widely used for monitoring CMV reactivation as well as the antigenemia assay that detects CMV structural phosphoprotein with a molecular weight of 65,000 (pp65). We developed a real-time PCR assay system for CMV based on a double-stranded DNA-specific dye, SYBR Green I, and quantified DNA, which was extracted automatically from plasma. This real-time PCR assay and the pp65 antigenemia assay were compared in parallel with 357 blood samples obtained from 64 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). Real-time PCR assay results correlated with those of the pp65 antigenemia assay (p < 0.0001). It is noteworthy that the detection of CMV DNA by PCR preceded the first positive antigenemia by 14 days. In this study, 10 of 64 patients developed CMV disease. The antigenemia assay detected CMV reactivation earlier than the development of CMV disease only in four of 10 patients. In contrast, our real-time PCR detected CMV-DNA before the development of CMV diseases in eight of 10 patients. The real-time PCR with SYBR Green I as a detection signal is simple and readily performed, and may be a useful system for early detection of CMV reactivation after allo-HSCT.     

Failure to detect human cytomegalovirus DNA in peripheral blood leukocytes of healthy blood donors by the polymerase chain reaction

The transmission of cytomegalovirus (CMV) by blood transfusion may have a major effect on certain immunocompromised patients. To protect susceptible blood recipients from infection, it is advisable to use blood components from CMV-seronegative donors. However, serologic tests are not capable of indicating which blood component actually harbors infectious virus and can transfer it to the recipient. Therefore, a sensitive method is needed for the detection of the virus itself. There have been three reports on the detection of CMV in healthy volunteer blood donors by the polymerase chain reaction (PCR). CMV DNA was found in all seropositive and most seronegative blood donors. However, many other authors have failed to confirm these data. A highly sensitive and specific PCR assay was developed for the detection of CMV DNA in peripheral blood leukocytes. With this protocol, blood samples from 116 volunteer blood donors were investigated. None of these samples proved to be positive for CMV DNA. In contrast, CMV DNA was detected in 10 of 10 renal transplant patients early in the course of active CMV infection. It can be concluded that the CMV genome copy number in the peripheral blood leukocytes of healthy individuals is beyond the detection limit of current PCR technology.     

Prenatal diagnosis of cytomegalovirus infection

Prognostic value of markers of cytomegalovirus infection (CMV) in pregnant women for the neonatal status was assessed. Detection of such markers as antiCMV IgM and CMV DNA in cervical secretion by DNA dot-spot hybridization in women with a complicated course of pregnancy indicates a 5.7% risk of delivery of children with stable symptoms. Studies of antibodies to pre-early proteins (IE CMV) showed that antiCMV IgG to IE are more incident in pregnant women than antiCMV IgM; moreover, antiCMV IgG to IE but not antiCMV IgM are detected in umbilical blood. The results of detection of antiCMV IgG and IgM to IE correlated with the clinical characteristics of newborns.     

A case of refractory cytomegalovirus-related thrombocytopenia that achieved complete remission without antiviral therapy

Cytomegalovirus (CMV) is one of the major infectious etiologies that induce thrombocytopenia. Although immune thrombocytopenic purpura (ITP) in children is often preceded by viral infections, thrombocytopenia associated with active CMV infection is considered CMV-related thrombocytopenia (CMV-thrombocytopenia), which can be distinguished from ITP. CMV-thrombocytopenia is reported to be less responsive to standard therapies for ITP and may require antiviral therapies. We herein report a case of refractory CMV-thrombocytopenia that achieved complete remission without antiviral therapy.A 20-month-old boy presented with a 2-day history of fever and systemic petechiae. There were no abnormal findings except for an extremely low platelet count (8000/μl) on blood examinations. He was clinically diagnosed with ITP, and intravenous immunoglobulin was administered twice, but his platelet count did not increase. CMV infection was suspected serologically, and a high CMV DNA load was detected in serum by real-time quantitative polymerase chain reaction (PCR). Without antiviral treatment, the CMV DNA load decreased below the detection limit on the 11th day of admission, followed by complete remission of the thrombocytopenia. The present case suggests that spontaneous recovery of thrombocytopenia can be expected in immunocompetent patients with CMV-thrombocytopenia in whom decreased CMV DNA load is observed.     

巨细胞病毒感染的细胞微丝骨架重组异常

本研究探讨巨细胞病毒(CMV)感染对人胚成纤维细胞(HF)微丝骨架影响及与病毒复制状态的可能关系。用显微镜观察细胞形态,采用RT-PCR检测β-actin表达,Western-blot检测胞内肌动蛋白质含量,间接免疫荧光标记病毒即刻早期抗原(IE)与微丝骨架探针观察HF细胞内感染状况及微丝骨架变化。结果表明CMV感染率大于95%,IE抗原主要位于胞核。受染细胞变粗、变圆,甚至脱壁,呈时间依赖性。感染后72、96小时,受染细胞内β-actin表达呈渐进性下调,差异显著(P<0.05);96小时后胞内β-actin含量则为未感染组的(74.2±13.4)%,差异显著(P<0.05)。受染细胞内微丝排列紊乱,极性消失;细胞融合,荧光强度明显减弱。结论CMV引起细胞内肌动蛋白质、微丝骨架形态及重组异常可能有助于其侵袭宿主细胞并进一步活化及复制。