造血干细胞移植中人巨细胞病毒检测：荧光定量聚合酶链反应的早期诊断价值

背景：动态监测造血干细胞移植受者血浆人巨细胞病毒载量,快速筛选出早期活动性人巨细胞病毒感染者,进而指导临床用药,提高造血干细胞移植的成活率。目的：探讨荧光定量聚合酶链反应对造血干细胞移植患者人巨细胞病毒活动性感染的早期诊断价值。方法：收集41例造血干细胞移植患者共656份血标本,应用实时荧光定量PCR方法动态监测血浆人巨细胞病毒DNA载量,并与60例健康体检者血浆人巨细胞病毒DNA载量对比分析。结果与结论：656份血标本中96份阳性,病毒载量介于5.0×10^2-1.0×10^7 IU/mL之间。治疗有效者人巨细胞病毒DNA迅速转阴,治疗耐药者持续阳性,12例连续阳性者2例死于巨细胞病毒活动性感染。实时荧光定量PCR方法检测人巨细胞病毒DNA适用于造血干细胞移植后巨细胞病毒活动性感染的早期诊断。     

HCG试纸条在HCG定量中的应用

人类巨细胞病毒（HCMV）在人群中感染率较高，是先天性感染中最常见的致畸致残的病毒。临床确诊比较困难。我们设计合成巨细胞病毒（HCMV）荧光定量PCR（FluorescenceQuantitativePCR，FQ－PCQ）诊断试剂盒，以一完全闭管式的PCR和荧光杂交技术相结合所产生的实时检测定量PCR方法，检测了３０１例产妇的血标本，３２４例新生儿脐血标本和２５６例儿童血标本HCMV－DNA的含量，同时与ELISA方法和常规PCR进行比较，探讨该方法在巨细胞病毒的临床诊断中的应用价值。３０１例产妇HCMV－IgM阳性４５例，阳性率为１４．９％，F…     

巢式PCR检测外周血血浆和白细胞中人巨细胞病毒核酸

目的比较用巢式PCR检测外周血血浆和白细胞中人巨细胞病毒（HCMV）DNA的阳性率。方法93例外周血标本分离血浆和白细胞，分别提取DNA后进行巢式PCR检测，同时进行荧光定量PCR以及HCMV IgM测定。结果白细胞HCM VDNA检出率为32．3％，明显高于血浆的16．1％（P〈0．017），巢式PCR检测敏感度高于荧光定量PCR（检出率8．6%，P〈0．017）；9例具有系列标本的患者中，用白细胞DNA检测的阳性例数和频度均高于血浆DNA。结论检测HCMV DNA时，以白细胞作为检测材料的阳性率高于血浆，巢式PCR敏感度高于荧光定量PCR。     

尿液上皮细胞中巨细胞病毒载量预测婴儿巨细胞病毒激活感染的价值

目的 探讨尿液上皮细胞(EC)中人巨细胞病毒(HCMV)DNA载量预测婴儿活动性HCMV感染的应用价值.方法 分别收集82例HCMV潜伏感染组和84例活动性感染组婴儿外周血和尿液标本,采用实时荧光定量聚合酶链反应(FQ-PCB)和化学发光免疫分析检测血浆HCMV DNA载量和HCMV IgM/IgG抗体;间接免疫荧光法检测外周血多形核白细胞(PMNLs)中HCMV pp65抗原;UF-100尿沉渣全自动分析仪和FQ-PCR分别做尿液EC计数和HCMV DNA载量检测,并计算尿液EC中HCMV DNA载量.同时,用受试者工作特征曲线(ROC)评价尿液上皮细胞中HCMV DNA载量在婴儿HCMV激活感染诊断中的敏感度和特异度.结果 166例HCMV感染婴儿尿液上皮细胞中HCMV DNA阳性检出率最高,为94.58%(157/166),病毒载量范围为5.67×102-1.31×107拷贝/103EC;不同时段尿液EC中HCMV DNA载量差异无统计学意义(F=0.19,P>0.05);活动性感染组尿液EC中HCMV DNA载量[5.13±0.99(拷贝/103EC,lg)]显著高于潜伏感染组[3.92±0.82(拷贝/103EC,lg),t=8.52,P<0.01];根据ROC曲线,当cut-off值为4.55时,尿液EC中HCMV DNA载量对活动性感染诊断的敏感度和特异度分别为71.4%和75.2%;活动性感染患儿更昔洛韦治疗后尿液EC中HCMV DNA载量显著低于治疗前(t=5.44,P<0.01).结论 尿液EC中HCMV DNA载量用于预测婴儿HCMV活动性感染是一种简便、有效的方法,并能用于治疗监测.     

荧光定量聚合酶链反应检测移植受者的巨细胞病毒DNA载量

目的评价实时荧光定量聚合酶链反应(PCR)检测移植后受者的巨细胞病毒感染情况。方法应用定性PCR检测59例骨髓移植受者和9例肝移植受者的150份外周血标本中的巨细胞病毒DNA,比较外周血白细胞和血浆的检出差异;应用实时荧光定量PCR检测上述150份血浆样本和54份对照血浆中巨细胞病毒DNA载量。结果以实时荧光定量PCR为参照,定性PCR检测白细胞的敏感度和特异度分别为62·5%和95·5%;检测血浆的敏感度和特异度分别为57·5%和99·1%。所检测的150份血浆样本中,定量PCR阳性率为26·7%,阳性者其病毒拷贝数介于5·065×102~3·570×105/ml之间;对照组仅1例阳性,且拷贝数小于103/ml。临床调查显示病毒拷贝数大于5×103的患者具有较高的机会发展为巨细胞病毒病。结论实时荧光定量PCR是一个快速、敏感、特异的,可用于监测移植后患者巨细胞病毒感染的方法,帮助临床及早治疗,缩短治疗时间。     

荧光定量聚合酶链反应检测巨细胞病毒DNA及其临床应用

人类巨细胞病毒(HCMV)在人群中感染率较高,是先天性感染中最常见的致畸致残的病毒.临床确诊比较困难.我们设计合成巨细胞病毒(HCMV)荧光定量PCR(Fluorescence Quantitative PCR,FQ-PCQ)诊断试剂盒,以一完全闭管式的PCR和荧光杂交技术相结合所产生的实时检测定量PCR方法,检测了301例产妇的血标本,324例新生儿脐血标本和256例儿童血标本HCMV-DNA的含量,同时与ELISA方法和常规PCR进行比较,探讨该方法在巨细胞病毒的临床诊断中的应用价值.     

实时荧光定量PCR技术在肾移植术后巨细胞病毒性肺炎诊断中的应用

目的探讨实时荧光定量PCR检测巨细胞病毒在肾移植术后肺部感染诊断中的应用价值。方法应用实时荧光定量PCR检测2002年1月至2006年1月在本院行肾移植术后肺部感染患者39例,健康自愿者50人的血清CMV DNA载量,应用SPSS10 0软件对检测结果进行统计学分析。结果肺部感染患者组39份样本中,19份可以检测到数值(阳性率为49%),其病毒拷贝数值介于1.05×103~4.26×105之间,平均为2.23×104;对照组中仅一份检测到数值(阳性率为2%),且CMV DNA小于104,其余49份均低于检测下限,实时荧光定量PCR检测CMV结果对病情发展有预测价值。结论实时荧光定量PCR技术为肾移植术后巨细胞病毒性肺炎的早期诊断提供了有效的试验手段,从而大大提高了肾移植术后巨细胞病毒性肺炎的治愈率。     

血液肿瘤患者血浆EB病毒和巨细胞病毒定量检测的临床价值

目的采用实时荧光定量聚合酶链反应(PCR)测定血液肿瘤患者血浆中EB病毒(EBV)和巨细胞病毒(CMV)DNA水平,探讨血浆EBV DNA和CMV DNA定量测定的临床意义。方法采用实时荧光定量PCR检测280例血液肿瘤患者血液中EBV DNA和CMV DNA载量。结果 280例血液患者中EBV DNA检测阳性率为17.1%,CMV检测阳性率为18.9%,动态监测病毒DNA拷贝数为10~2~10~7 copy/mL。接受外周造血干细胞移植的患者126例,其中EBV检测阳性率为28.6%,CMV检测阳性率为40.5%。血液肿瘤患者进行抗病毒治疗后,均在7~14d后转阴。结论实时荧光定量PCR动态检测血液肿瘤患者EBV DNA和CMV DNA水平对于EBV和CMV感染患者的疗效判断具有重要临床意义。     

荧光定量PCR快速检测婴儿中枢神经系统巨细胞病毒

目的探讨荧光定量PCR技术快速诊断中枢神经系统损伤婴儿人巨细胞病毒(HCMV)感染的临床价值,为婴儿HCMV先天性感染的快速特异性诊断提供实验依据。方法分别用实时荧光定量PCR技术和ELISA法检测84例疑似先天性HCMV感染的中枢神经系统损伤婴儿中尿液HCMV-DNA及血清HCMV-IgM。结果 84例中枢神经损伤婴儿尿液中,29例HCMV-DNA阳性,检出率为34.5%。活动性HCMV感染病毒载量介于5.9×105～7.6×102 copy/mL。HCMV-IgM阳性15例,检出率17.8%。两种方法阳性检出率差异有统计学意义(χ2=6.87,P<0.01)。结论婴儿先天性HCMV感染是引起其中枢神经系统损伤的重要原因之一。在连续动态检测患儿HCMV多种指标时,实时荧光定量PCR法可快速、特异地定量检测HC-MV-DNA,有效反映体内HCMV的活动性感染,在HCMV感染的快速诊断、疗效观察方面具有更好的临床应用价值。     

巨细胞病毒定量PCR与pp65抗原测定监测异基因造血干细胞移植巨细胞病毒感染的比较

本研究比较巨细胞病毒（CMV）定量PCR检测和CMV—pp65抗原检测在异基因造血干细胞移植CMV感染中的诊断价值。以84例异基因造血干细胞移植患者为研究对象,自预处理开始每周对患者EDTA抗凝外周血血标本进行CMV—pp65抗原及cMV定量PCR动态检测,直至出院,比较观察两者在诊断CMV感染中的作用。结果表明：84例移植患者的732份系列血标本中,26例移植后检出CMV定量PCR阳性,检出率30．95％,其中9例为cMV血症,13例为cMV病,检出中位时间为37．1（7—105）天;22例CMVpp65抗原检测阳性,检出率26．19％,检出中位时间为46．6（10—128）天;所有CMV—pp65抗原检测阳性患者CMV定量PCR均为阳性,4例CMV定量PCR阳性但CMV—pp65抗原检测阴性患者均未发展为CMV病。CMV病多出现于CMV定量PCR中等至高拷贝数或中等至高水平病毒血症（CMV—pp65抗原）病例中。治疗后CMV定量PCR转阴中位时间为17．5（11—28）天。CMV—pp65抗原转阴中位时间为10．0（7—21）天。结论：CMV定量PCR和CMV—pv65抗原检测均能作为异基因造血干细胞移植CMV感染早期诊断的有效手段,CMV定量PCR更敏感,CMV—pp65抗原检测更特异,两者同时使用能更有效地提高CMV感染诊断水平,监控其发生发展。     

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.     

基于核酸序列的扩增技术在异基因外周血干细胞移植后受者HCMV感染诊断的应用

本研究探讨基于核酸序列的扩增技术（nucleic acid sequence based amplification, NASBA）检测mRNA对异基因外周血干细胞移植受者术后人类巨细胞病毒（HCMV）感染的诊断价值，并评价该技术对抗病毒治疗的指导意义。以128例移植术后患者为研究对象，采集移植术后外周血352份；利用NASBA检测外周血中HCMV基质外壳蛋白PP67（UL65）mRNA，并与HCMV DNA的结果进行比较。结果表明：352份血标本中，HCMVmRNA阳性105份（29．83％），HCMV DNA阳性183份（51．99％）。128例患者术后有45例发展为HCMV病，HCMV DNA和HCMV mRNA检测诊断HCMV病的敏感性和特异性分别为95.56％（43／45）、93.33％（42／45）和60．24％（50／83）、97．59％（81／83）。两者特异性的比较差异具有显著性（P〈0、05）。结论：HCMV-PP67的NASBA检测方法具有快速、敏感性高和特异性强等优点，可作为快速检测HCMV病的方法用于临床。该法检测结果与移植受者的临床症状相关。故也可用作监视异基因造血干细胞移植受者术后HCMV感染和指导抗病毒治疗的依据。     

婴儿肝炎综合征中人类巨细胞病毒感染检测方法的比较

目的：探讨婴儿肝炎综合征中人类巨细胞病毒(HCMV)感染的检测方法。方法：用HCMV pp65抗原血症法及荧光定量聚会酶链反应(FQ-PCR)法检测24例患儿34份血浆及多形核白细胞(PMNLs)标本。结果：HCMV pp65抗原血症法诊断巨细胞病毒感染所致婴儿肝炎综合征的特异度为100％，灵敏度为90％；FQ-PCR法检测PNNLs的特异度为100％，灵敏度为60％；血浆FQ-PCR法的特异度为100％，灵敏度为50％。同一份血标本其血浆中的HCMVDNA拷贝数低于PMNLs，使得血浆FQ-PCR法在HCMV诊断及疗效观察方面具有滞后现象。动态观察发现HCMV pp65抗原血症法与FQ-PCR法检测的PMNLs结果与婴儿肝炎综合征患儿的治疗过程有较好的符合率。结论：HCMV pp65抗原阳性与HCMV活动性感染密切相关，可作为婴儿肝炎综合征患儿疗效的观察指标。HCMV pp65抗原血症法与FQ-PCR法可作为临床诊断HCMV症状性感染的一种快速、有效的实验室方法。     

Development of multiplexed real-time quantitative polymerase chain reaction assay for detecting human adenoviruses

Adenoviruses (AdVs) have been associated with a wide variety of human disease and are increasingly recognized as viral pathogens that can cause significant morbidity and mortality in immunocompromised patients. Early detection of AdV DNA in plasma and sterile fluids has been shown to be useful for identifying patients at risk for invasive AdV disease. Because of the large number of existing Adv types, few real-time quantitative AdV polymerase chain reaction (PCR) assays published effectively cover all AdV types. We designed a series of AdV PCR primers and probes and empirically multiplexed them into 2 separate real-time PCR assays to quantitatively detect all 49 serotypes of human AdV (types 1–49) available from American Type Culture Collection. We then subsequently multiplexed all the primers and probes into 1 reaction. The sensitivity of these assays was determined to be less than 10 copies per reaction (500 copies/mL plasma). In a retrospective evaluation, we detected all 84 clinical AdV isolates isolated in cell culture from patients undergoing hematopoietic stem cell transplantation between 1981 and 1987. Prospective analysis of 46 consecutive clinical samples submitted for AdV testing showed greater sensitivity and equal specificity of the AdV PCR than viral culture. This real-time PCR assay allows rapid, sensitive, and specific quantification of all currently defined AdVs into either 2 or 1 multiplex assay for clinical samples.     

Quantitative real-time PCR with automated sample preparation for diagnosis and monitoring of cytomegalovirus infection in bone marrow transplant patients

BACKGROUND: In bone marrow and stem cell transplant patients, the widespread use of preemptive cytomegalovirus (CMV) antiviral therapy necessitates faster, more precise, and more sensitive quantitative laboratory methods for serial viral load monitoring. METHODS: We developed a novel CMV viral load assay using real-time PCR of plasma DNA prepared by an automated robotic workstation. Fluorescent hybridization probes directed at the glycoprotein B (gB) gene (or EcoRI D region) of CMV were used to detect and quantify PCR products. The beta-globin gene was amplified in parallel to control for the efficiency of the extraction and PCR steps. RESULTS: The assay was linear (R = 0.999) from a lower detection limit of 125 copies/mL to 5 x 10(9) copies/mL with a PCR efficiency of 1.975 (gB) or 2.02 (EcoRI D). The viral loads determined by PCRs directed at these two different viral targets were no different (n = 53; R = 0.928). The interassay CV was 3.5%, and the intraassay CV was 1-4%. Compared with a commercially available quantitative competitive PCR assay (Roche MONITOR; R = 0.59), the mean CMV viral load by real-time PCR was 3.1 times higher (mean ratio; P = 0.002). The diagnostic sensitivity and specificity of the real-time assay were 96% and 100%, respectively (n = 147), compared with 74% and 98% for a qualitative PCR assay (Roche AMPLICOR). On a subset of samples, the diagnostic sensitivity of viral culture was no greater than 50% (n = 44). Of 1115 clinical referral samples from 252 patients, 10% of the samples and 18% of the patients had low-level CMV viremia (median, 500 copies/mL). In this predominantly (85%) bone marrow transplant testing cohort, serial CMV viral load results were the predominant clinical trigger for the initiation, monitoring, and cessation of preemptive antiviral therapy. CONCLUSIONS: The combination of automated DNA preparation and semiautomated real-time fluorescent PCR detection allows for a sensitive, precise, and accurate high-throughput assay of CMV viral load that can be used as the laboratory trigger for preemptive antiviral therapy.     

尿BK病毒载量与干细胞移植后出血性膀胱炎的关系

目的 分析BK病毒尿与造血干细胞移植(HSCT)术后迟发性出血性膀胱炎(LOHC)的相关性,探讨BK病毒载量与LOHC发病的关系.方法 收集2006年8月至2008年4月113例HSCT患者的晨尿标本,提取病毒DNA,崩PCR方法扩增多瘤病毒DNA,以4JD名健康成人为对照.用实时定量PCR方法检测尿BK病毒阳性患者在LOHC不同时期的尿标本中BK病毒DNA拷贝数.结果 113例HSCT患者中22例(19.5%)发生HC,中位发生时间为+44(+13～+114)d,包括1级7例,2级11例,3级3例,4级1例.其中21例(95.5%)LOHC患者尿标本经PCR检出BK病毒,检出率明显高于非LOHC组(31.9%)(P=0.000).健康对照组均未检出BK病毒.定量PCR检测结果显示,LOHC患者尿BK病毒DNA拷贝数在HC发病前1周的平均水平较初次阳性时有明显上升(105拷贝数/μl比104拷贝数/μl),在HC发病当周及缓解后1周无明显改变.不同级别LOHC患者在发病前和发病时尿BK病毒DNA拷贝数差异无统计学意义.非LOHC患者尿中BK病毒DNA拷贝数平均水平为103～104拷贝数/μl,低于LOHC患者.结论 BK病毒尿是HSCT后LOHC的重要致病原因.如HSCT患者尿中BK病毒DNA拷贝数呈动态上升并超过105拷贝数/μl时可能预示LOHC的发生.     

造血干细胞移植后BK病毒监测的临床意义

本研究旨在建立造血干细胞移植后BK病毒(BKV)感染的快速检测方法,探讨该方法的可行性和应用价值。根据BKV基因序列设计引物,构建用于检测的定量标准品,建立实时荧光定量PCR检测BKV的方法,并对36例造血干细胞移植术后患者的尿液标本中BKV含量进行检测。结果表明:成功构建了BKV的定量标准品和荧光定量PCR检测BKV的方法,并经过特异性、敏感性、稳定性和重复性验证;在36例造血干细胞移植患者中有20例尿液定量检测出BKV,阳性率为55.56%,尿液负荷量BKV拷贝数为2.46×104-7.8×109/ml。结论:BKV实时荧光定量PCR检测方法的建立对造血干细胞移植后出血性膀胱炎的早期诊断、预防、治疗具有重要意义。     

HCMVpp65抗原和IgM抗体检测诊断儿童HCMV活动性感染的比较

目的：比较人巨细胞病毒(HCMV)pp65抗原(以下简称pp65)检测和血清HCMV-IgM抗体(以下简称IgM抗体)检测两种方法对儿童HCMV活动性感染的诊断实用意义。方法：采集临床疑似HCMV活动性感染儿童血标本(共251份)，分离血浆和多形核白细胞，分别用于IgM抗体检测和pp65检测。同时进行荧光定量PCR检测HCMVDNA，与pp65抗原作平行比较。结果：pp65抗原检测的结果与IgM抗体检测的符合率为73.3％。与HCMV DNA检测相比pp65抗原检测法的符合率、特异度和敏感度分别为85．5％，85．2％和86．2％。而且高pp65抗原血症与患者的临床症状密切相关。结论：pp65抗原血症反映该病毒活动状况，可监测HCMV活动性感染。由于儿童患者还存在原发感染的可能性，所以．联合HCMV-IgM的监测来提高临床的诊断率。     

Comparison of quantitative competitive polymerase chain reaction-enzyme-linked immunosorbent assay with LightCycler-based polymerase chain reaction for measuring cytomegalovirus DNA in patients after hematopoietic stem cell transplantation

Development of highly sensitive quantitative assays for cytomegalovirus (CMV) DNA detection is crucial for identification of immunodeficient patients at high risk of CMV disease. We designed 2 internally controlled competitive quantitative assays, enzyme-linked immunosorbent assay (ELISA)-based and real-time polymerase chain reaction (PCR) tests, using amplification of the same segment of the CMV genome. The aim of this study was to compare sensitivity, specificity, and laboratory performance characteristics of these assays. In both assays, a 159-bp segment of UL83 gene was amplified. External and internal controls were constructed by cloning the amplification product and heterogenous DNA segment flanked by target sequences for CMV-derived primers into bacterial plasmids, respectively. Real-time PCR was performed on LightCycler (Roche Diagnostics, Mannheim, Germany), and amplicons were detected using fluorescence resonance energy transfer probes. Alternatively, PCR products were labeled by digoxigenin, hybridized to immobilized probes, and detected by ELISA. The assays were tested on genomic DNA isolated from laboratory strains of CMV, QCMD control panel, and CMV DNA-positive peripheral blood DNA samples from hematopoietic stem cell transplant recipients, previously characterized by pp65 antigenemia and qualitative nested PCR. Real-time and ELISA-based PCR assays showed a linear course of 1-10(8) and 10-10(5) copies of CMV DNA per reaction, respectively. When compared with ELISA-based PCR, real-time PCR showed superiority in inter- and intra-assay reproducibility. Both assays were highly specific in detecting CMV DNA. No difference in amplification efficiency of internal or external standards and wild-type CMV DNA was found. The assays exhibited 83% concordance in CMV DNA detection from clinical samples, all discrepant samples having low CMV DNA copy numbers. There was a good correlation between viral DNA loads measured by the 2 assays. Statistically significant correlation was observed between the numbers of CMV DNA copies and pp65-positive leukocytes in the samples tested. Both variants of competitive PCR are adequately sensitive to be used for CMV DNA quantitation in clinical samples. LightCycler PCR, having superior performance characteristics and being less time-consuming, seems to be more suitable for routine diagnosis.