PCR技术和血清抗体检测妊娠期巨细胞病毒感染价值的比较

将ＰＣＲ技术检测孕妇血白细胞中ＨＣＭＶ－ＤＮＡ和ＥＬＩＳＡ检测孕妇血清中ＨＣＭＶ特生ＩｇＭ和ＩｇＧ进行对比研究。结果：ＤＮＡＰＣＲ阳性率为４２．００％，与ＩｇＭ阳性率２５．５８％比较，Ｐ〈０．０５，与ＩｇＧ阳性率６２．７９％比较，Ｐ〈０．０１，说明ＤＮＡＰＣＲ较ＩｇＭ检测敏感，但不如ＩｇＧ。结论：ＤＮＡＰＣＲ更适用于论断先天性ＨＣＭＶ感染和一部分检测不出ＩｇＭ的ＣＭＶ复发感染者，对妊娠期ＣＭＶ感染     

石家庄地区新生儿尿巨细胞病毒感染的临床分析

采用聚合的酶链反应（ＰＣＲ）检测１０５例出生后３日内新生儿尿中巨细胞病毒,同时应用酶联免疫吸附法（ＥＬＩＳＡ）检测脐血特异性ＩｇＧ和ＩｇＭ抗体。结果,新生儿尿液ＨＣＭＶ－ＤＮＡ阳性者３３例,阳性率３１．４％,脐血ＩｇＧ抗体阳性率１００％,ＩｇＭ挤体阳性率１．９％。结果表明ＰＣＲ技术检测新生儿尿液ＨＣＭＶ－ＤＮＡ有利于ＨＣＭＶ先天性感染的早期诊断,早预防、早阻断。有利于更好地实行计划生育,有利于优生     

巨细胞病毒宫内感染的诊断及对胎儿的影响

采用地高辛探针斑点杂交技术和聚合酶链反应（ＰＣＲ）技术，对５１例人巨细胞病毒（ＨＣＭＶ）血清学抗体ＩｇＭ阳性孕妇的羊水、脐血和产后两周内的新生儿尿液，进行ＨＣＭＶＤＮＡ检测。同时与酶联免疫吸附法（ＥＬＩＳＡ）检测脐血、羊水中ＨＣＭＶ－ＩｇＭ的结果相比较。结果，地高辛探针斑点杂交检测ＨＣＭＶ－ＤＮＡ的阳性率分别为：羊水２１．５７％，脐血３３．３３％，新生儿尿液２７．４５％。ＰＣＲ检测ＨＣＭＶ－ＤＮＡ的阳性率分别为：羊水２９．４１％，脐血４３．１４％，新生儿尿液３Ｓ，２９％。羊水ＨＣＭＶ－ＩｇＭ阳性率为１１．７６％，脐血ＨＣＭＶ－ＩｇＭ阳性率为２３．５３％。脐血ＨＣＭＶ－ＤＮＡ阳性的新生儿平均出生体重明显低于脐血ＨＣＭＶ－ＤＮＡ阴性的新生儿，而脐血ＨＤＷ－ＤＮＡ阳性的新生儿肝功能异常、血小板减少以及低Ａｐｇａｒ评分的发生率明显高于脐血ＨＣＭＶ－ＤＮＡ阴性的新生儿。结果表明：采用ＥＲ技术检测羊水、脐血或产后两周内新生儿尿液ＨＣＭＶＤＮＡ有助于ＨＣＭＶ官内感染的早期诊断，便于临床早期干预，ＨＣＭＶ官内感染严重影响胎儿的生长发育，可造成胎儿肝功能异常、血小板减少和新生儿窒息的发生。     

产科领域内TORCH感染的初步临床研究

调查本地区妊娠妇女ＴＯＲＣＨ感染的发生率以及母婴垂直传播发生情况，从而指导优生优育。方法：采用ＥＬＩＳＡ法，对我院８８２例孕中期或晚期孕妇外周血血清和相应产妇脐血血清行ＴＯＲＣＨ系列抗体（ＩｇＧ、ＩｇＭ）筛查。结果：ＴＯＲＣＨ系列特异性抗体阳性１９５例，阳性率为２２．１％；其中单项病毒抗体阳性９３例，双项阳性５５例，三项阳性３７例，四项阳性者１０例；ＴＯＲＣＨ系列ＩｇＧ阳性１０７例，ＩｇＭ阳性１２８例，ＩｇＧ、ＩｇＭ均阳性１０９例。脐血特异性抗体ＩｇＧ阳性１１６例，ＩｇＭ阳性１１例，其垂直传播率ＣＭＶ５０％、ＲＵＶ２４．７％、ＴＯＸ３１．８％、ＨＳＶ２３３．３％。感染组与未感染组比较对胎婴儿影响极大，特别是胎儿生长发育、围产儿死亡以及对ＩｇＭ阳性病例复采用ＰＣＲ测定法，进一步确诊。对ＴＯＲＣＨ阳性患者应采取相应措施     

检测HBVDNA/Ig-双特异性循环免疫复合物的免疫捕捉法PCR

目的;建立一种免疫捕捉法ＰＣＲ技术,研究血清免疫复合物中不同Ｉｇ类型抗体结合ＨＢＶ的情况,并初步探讨其意义。方法：以羊抗人μ,γ,α链的ＩｇＧ为捕捉抗体,再利用ＰＣＲ扩增捕捉物中的ＨＢＶＤＮＡ。结果;成功地建立了检测ＨＢＶＤＮＡ／ＩｇＭ,ＩｇＧ和ＩｇＡ－ＴＣＩＣ的免疫捕捉法ＰＣＲ技术。     

用重组结构区抗原检测抗丙型肝炎病毒IgM抗体方法的建立

目的检测抗丙型肝炎病毒（ＨＣＶ）结构区蛋白ＩｇＭ抗体。方法采用丙型肝炎病毒Ｃ，Ｅ１，Ｅ２区重组抗原混合包被和分别包被酶标板；用兔抗人γ链血清处理人血清标本，再用固相包被羊抗兔抗体吸附兔抗人γ链－人ＩｇＧ复合物，建立了抗－ＨＣＶＩｇＭ检测方法。结果对７６例丙型肝炎病人血清进行抗－ＨＣＶＩｇＭ检测，同时与逆转录－巢式聚合酶链反应（ＲＴ－ＰＣＲ）检测结果进行比较，两者具有相关性（Ｐ＜０００５）；ＩｇＭ抗体组成分析结果表明采用全片段Ｃ、Ｃ＋Ｅ２区抗原血清标本中的抗－ＨＣＶＩｇＭ检出率分别可达９６６％和１００％。结论ＨＣＶ的Ｃ、Ｅ２重组抗原用于抗－ＨＣＶＩｇＭ检测具有较高的敏感度和特异性。     

用聚合酶链反应行巨细胞病毒感染产前诊断的研究

对５０例正常育龄妇女、６３例正常孕妇及２０例有异常孕产史的孕妇进行了血清巨细胞病毒抗体ＣＭＶ－ＩｇＧ、ＣＭＶ－ＩｇＭ的检测及聚合酶链反应（ＰＣＲ）的检测，并对正常孕妇及异常孕产史孕妇进行绒毛ＣＭＶＰＣＲ检测。结果正常孕妇血清ＣＭＶＰＣＲ阳性率为３１．７５％，远远高于正常非孕妇的血清ＣＭＶＰＣＲ的阳性率１６％。正常孕妇人流的绒毛ＣＭＶＰＣＲ阳性率为２０．６３％，明显低于有异常孕产史孕妇绒毛ＰＣＲ的阳性率４５％，且异常孕产史孕妇血清ＣＭＶＰＣＲ阳性率明显高于正常孕妇。另对血清ＰＣＲ阳性的１２例早孕时抽取绒毛测ＰＣＲ，阳性率为７５％（９／１２），提示宫内感染率相当高。对正常和异常孕产史孕妇血清ＩｇＭ与ＰＣＲ比较，后者敏感性远超过前者，用ＰＣＲ方法对早孕期ＣＭＶ感染能比较正确地早期作出宫内感染的诊断。     

成都地区孕妇和新生TORCH感染的检测与分析

采用ＥＬＩＳＡ检测孕妇血清及新生儿脐血ＴＯＲＣＨ特异性ＩｇＧ，ＩｇＭ抗体，同时采用ＰＣＲ检测ＣＭＶ－ＩｇＭ阳性孕妇的羊水及产后乳汁ＣＭＶ－ＤＮＡ，以了解孕妇及新生儿ＴＯＲＣＨ感染情况。结果显示ＴＯ，ＲＶ，ＣＭＶ，ＨＳＶ－１和ＨＳＶ－２检出率分别为４１．４８％，８９．３６％，９５．７４％，３９．３６％和２２．３４％，其活动性感染分别为４．２５％，３．１９％，２４．４６％，１１．７０％和５．３１％，新     

用重组结构区抗原检测抗丙型肝炎病毒IgM抗体方法？…

目的 检测抗丙型肝炎病毒（ＨＣＶ）结构区蛋白ＩｇＭ抗体。方法 采用丙型肝炎病毒Ｃ，Ｅ１、Ｅ２区重组抗原混合包被和分别包被酶标板；用兔抗人γ链血清处理人血清标本，再用固相包被羊抗兔抗体吸附兔抗人γ链－人ＩｇＧ复合物，建立了抗－ＨＣＶＩｇＭ检测方法。结果 对７６例现型肝炎病人血清进行抗－ＨＣＶ ＩｇＭ检测，同时与逆转录－巢式聚合酶链反应（ＲＴ＋ＰＣＲ）检测结果进行比较，再会得具有相关性（Ｐ〈０．００５     

人巨细胞病毒感染对肾移植受者血清sIL－2R水平的影响

用ＰＣＲ检测ＨＣＭＶ－ＤＮＡ，ＥＬＩＳＡ法检测ＨＣＭＶ－ＩｇＭ及ＩｇＧ，以诊断肾移植受者ＨＣＭＶ感染。用双抗体夹心法ＥＬＩＳＡ检测６５例肾移植受者血清ｓＩＬ－２Ｒ水平，结果表明：ＨＣＭＶ感染后宿主血清ｓＩＬ－２Ｒ水平明显增高（Ｐ＜０．０１），且ＨＣＭＶ疾病组ｓＩＬ－２Ｒ增高程度大于无症状感染组（Ｐ＜０．０１）；６例原发性ＨＣＭＶ感染者ｓＩＬ－２Ｒ水平与ＩｇＭ水平呈正相关（ｒ＝０．９９０８），提示随感染程度增加，血清ｓＩＬ－２Ｒ水平随之增高，还发现血清ｓＩＬ－２Ｒ水平与Ｃ９４／ＣＤ８比值是负相关（ｒ＝－０．９７８９），说明ＨＣＭｖ感染后ｓＩＬ－２Ｒ水平增高与Ｔ细胞亚群改变有关，反之也说明ｓＩＬ－２Ｒ增高程度可表明体内免疫抑制状态。对于ＨＣＭＶ感染后血清ｓＩＬ－２Ｒ水平增高的机理有待进一步探讨。     

Diagnosis of cytomegalovirus infection by detection of the early antigen of cytomegalovirus in cell cultures

Conventional virus isolation and detection of cytomegalovirus (CMV) early antigen by immunofluorescence staining of cultured cells were compared in the diagnosis of CMV infection from urine specimens. By virus isolation, 33 specimens out of 333 studied were positive, and the mean length of culturing time for a positive result was 23 days (range from 6 to 45 days). By early antigen detection, 35 specimens were positive after 20 hours in culture, but the number of positive findings increased to as high as 49 after 7 days in culture. It is recommended that, in addition to the early antigen staining after one day in culture, cells should also be stained after one week in culture, because the sensitivity is essentially improved by extended culturing time.     

Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen.

A commercially available monoclonal antibody directed against an early nuclear protein of cytomegalovirus was used with low-speed centrifugation for the rapid detection of this virus from urine specimens inoculated onto MRC-5 cells. A total of 19 of 162 (11.7%) urine specimens inoculated were positive by both immunofluorescence and peroxidase-antiperoxidase procedures (sensitivity, 100%), whereas only 18 of the samples produced cytopathic effects in conventional cell culture (specificity, 94.7%). All specimens were positive by immunofluorescence and peroxidase-antiperoxidase procedures at 36 h postinfection, whereas an average of 9 days was required for cytopathic effects to develop in cell cultures.     

Dot immunoperoxidase assay using monoclonal antibody for direct detection of cytomegalovirus in urine samples.

A rapid, simple dot immunoperoxidase assay for the direct detection of cytomegalovirus in clinical urine samples was developed. The assay was performed on nitrocellulose paper dotted with urine pellets free of cellular debris. Cytomegalovirus was detected with a monoclonal antibody to the capsid antigen, and the complex was visualized by immunoperoxidase staining. Positive reactions appeared as well-defined dark blue spots. Of the 87 urine samples examined, 10 proved positive in the dot immunoperoxidase assay, and 77 proved negative. The results agreed completely with the detection of cytomegalovirus-induced antigens in cell cultures inoculated with clinical specimens.     

Comparison of MRC-5 and continuous cell lines for detection of cytomegalovirus in centrifugation cultures.

Continuous cell lines were assessed for use for rapid human cytomegalovirus (HCMV) detection procedures combining tissue culture, centrifugation, and immediate early antigen (IEA) immunostaining. Human cells (MRC-5 embryonic fibroblasts, U-373MG astrocytoma cells, differentiated teratocarcinoma (Tera-2) cells), murine cells (BALB/c-3T3 and Y-1 cells), BHK21 hamster cells, and mink lung (ML) cells were first inoculated with HCMV laboratory strain. IEA synthesizing cells were detected by immunoperoxidase assay using a monoclonal antibody. ML cells and differentiated Tera-2 cells exhibited more positive cells than MRC-5 cells. BHK21, and MRC-5 cells were equivalent in sensitivity whereas U-373MG, BALB/c-3T3, and Y-1 cells had only reduced IEA positive cells. When 63 urine specimens were inoculated onto MRC-5, ML and differentiated Tera-2 cells, 20 (31.7%) were positive in MRC-5 cells versus 18 (28.5%) in ML or Tera-2 cells. Moreover, greater numbers of infected cells were detected in MRC-5 cells than in these two cell lines. MRC-5 cells were superior for detection of HCMV in clinical samples by centrifugation cultures.     

Early detection of cytomegalovirus in cell culture by a monoclonal antibody

A commercially available monoclonal antibody directed against early cytomegalovirus (CMV) antigen was used for the demonstration of CMV by immunofluorescence (IF) in cell culture within 2 days. The results were compared with the appearance of CMV-specific cytopathogenic effect (CPE). Urine specimens from 31 healthy children in day-care centers were inoculated on human embryonic fibroblasts. In addition, 45 CMV strains that had been stored at -70 degrees C were reinoculated. CMV was detected in 8/31 urine specimens by IF and 7 of these gave a specific CPE at an average of 16 days post-inoculation. One specimen was negative by IF but specific CPE was found at day 13. After reinoculation, CMV was detected in 76% by IF while 44 specimens developed CPE within a 6-week period. Demonstration of early CMV antigen in cell culture was found to be a rapid method for early diagnosis of CMV. Since the conventional cell culture with detection of CPE was more sensitive it may be useful to combine the two methods.     

Rapid detection of cytomegalovirus by 24-well plate centrifugation with the use of a monoclonal antibody to an early nuclear antigen

Two methods for the detection of cytomegalovirus (CMV) in 457 clinical specimens were compared: (1) centrifugal inoculation of MRC-5 cells seeded on coverslips in 24-well plates and staining with a monoclonal antibody to CMV early nuclear antigen after incubation for both 16-18 hours (EA-1) and four days (EA-4); and (2) conventional tube cell culture. CMV was identified in 50 (11%) specimens from 34 different patients. EA-1 and EA-4 had positive results for CMV in 32 (64%) and 36 (73%) of the specimens, respectively. Positive inclusions were present on only one coverslip in 31% of the cases by EA-1 and in 10% by EA-4. The number of inclusions was not necessarily predictive of tissue culture results. CMV was recovered by conventional tissue culture from 27 specimens (54%) after an average of 17 days (range, 6-26 days). One specimen, positive for CMV by EA-4, yielded herpes simplex virus (HSV), and from 9 of the 407 CMV-negative specimens, another virus was recovered: HSV from 6 specimens and varicella zoster virus, adenovirus, and enterovirus from one specimen each. CMV was detected in significantly more specimens by EA-4 than by tissue culture (P = 0.037). However, there was no significant difference in the detection of CMV between EA-1 and EA-4 or between EA-1 and conventional culture. The authors' data suggest that for maximum recovery of CMV from clinical specimens, both an early antigen assay and conventional tissue culture should be performed. For urine specimens it appears that inoculation of two coverslips followed by staining after overnight incubation is adequate. To optimize the yield of the early antigen assay when testing specimens other than urine, the authors recommend inoculating three coverslips, two of which should be stained after overnight incubation, and, if necessary, the third coverslip could be stained after a more prolonged incubation period.     

Differential recovery of cytomegalovirus from cellular and supernatant components of bronchoalveolar lavage specimens.

The recovery of cytomegalovirus from bronchoalveolar lavage (BAL) specimens was compared after inoculation of MRC-5 tube and shell vial cell cultures with four different BAL preparations. Analysis of culture results obtained with 55 cytomegalovirus culture-positive samples showed significant differences in the ability to isolate virus from the supernatant and cellular components of these specimens. There was a 52% reduction in cytomegalovirus recovery and a significant delay in the development of cytopathic effect in cultures inoculated with the cellular component of BAL specimens when compared to cultures inoculated with crude BAL cells and fluid. The mean time for detection of cytopathic effect was 11.8 days in tubes inoculated with crude BAL and 18.2 days for tubes inoculated with BAL cells. A similar effect was observed using a rapid shell vial culture technique. A 39% reduction in the number of isolates and a 57% reduction in the number of positive cells were observed in vials inoculated with cells when compared to cultures inoculated with crude BAL. By contrast, using cell-free BAL supernatant as inoculum did not reduce the number of positive cultures or delay development of cytopathic effect. The results suggest that in most BAL specimens, cytomegalovirus is associated with the cell-free, rather than the cellular, component. Although BAL cell concentrates frequently are used for cultivation of viruses from BAL, our results showed that the use of these preparations results in a significant number of false-negative cytomegalovirus cultures.     

Sensitive non-isotopic DNA hybridisation assay or immediate-early antigen detection for rapid identification of human cytomegalovirus in urine.

A sensitive non-radioactive DNA hybridisation assay employing digoxigenin-labelled probes was compared with immediate-early antigen detection and conventional virus isolation for the identification of human cytomegalovirus (HCMV) in 249 urine samples. Of 44 specimens yielding HCMV by virus isolation, more were positive by DNA hybridisation (32; 73%) than by immediate-early antigen detection (25; 52%) (P = 0.05). The specificity of the hybridisation assay in 45 apparently falsely positive specimens was supported by detection of HCMV DNA in 40 of these specimens using the polymerase chain reaction. Many urine specimens may thus contain large amounts of non-viable virus or free viral DNA. Evaluation of various protocols for the extraction and denaturation of virus DNA prior to hybridisation showed that proteinase K digestion with phenol/chloroform extraction was the most sensitive and reliable procedure. We conclude that the non-radioactive DNA hybridisation assay described is a potentially valuable routine diagnostic test.     

Enhanced recovery of cytomegalovirus in conventional tube cultures with a spin-amplified adsorption.

Low-speed centrifugation-mediated adsorption was evaluated as an enhancement of infectivity of clinical and laboratory strains of cytomegalovirus (CMV) occurring with cells grown in conventional culture tubes. The time required for reporting of primary isolates of CMV from urine specimens adsorbed onto monolayers of WI-38 cells in culture tubes was calculated. Of 668 specimens adsorbed by the stationary phase (SP) method, 98 were positive by cytopathic effect (CPE) that required an average of 16.8 days for recovery in culture. However, the appearance of CPE required a shorter average time of 11.9 days for 70 CMV strains isolated from 283 specimens adsorbed in tube cultures by the spin-amplified (SA) method. In another phase of clinical CMV recovery, urine specimens were adsorbed by the SA method onto cell cultures grown in both shell vials and test tubes. Of 594 specimens inoculated, a total of 74 were positive by either CPE in test tubes or immunostaining-localized early antigen in shell vials. Approximately one-third of these CMV isolates were recovered only by CPE from specimens adsorbed by the SA method in test-tube cultures. In a related study to further evaluate differences between adsorption methods, the AD-169 laboratory strain of CMV was adsorbed by SP and SA methods onto MRC-5 cells grown in both culture vessels. Early antigen detection by immunomicroscopy was found in the infected cells at least 2 to 4 days prior to the appearance of CPE, regardless of adsorption procedure. In both vessels, the replication of AD-169 virus in cultures adsorbed by the SA method consistently exceeded that of virus adsorbed by the SP procedure. CPE occurred 24 to 48 h earlier and progressed two to four times more extensively; early antigen was expressed two- to fourfold greater within 24 to 48 h postinfection; and foci of infected cells containing late antigen were two to four times greater in number at 1, 2, and 5 days postinfection. Overall, the replication and enhancement of infectivity of laboratory and clinical strains of CMV as determined by CPE and early and late antigen expression occurred most efficiently with specimens adsorbed by the SA method onto cultures grown in conventional tubes or shell vials.     

Evaluation of number of shell vial cell cultures per clinical specimen for rapid diagnosis of cytomegalovirus infection.

Specimens submitted for the diagnosis of cytomegalovirus (CMV) infection were inoculated into three (blood) or two (urine, tissue, bronchoalveolar lavage [BAL]) shell vials seeded with MRC-5 cells for the diagnosis of CMV infection. We evaluated the detection of 993 specimens that were positive for CMV according to the number of shell vial cell cultures inoculated per specimen. For blood cultures, and considering one CMV-positive shell vial as 100%, inoculation of three shell vials versus one increased the detection rate of the virus by 51%. Inoculation of three shell vials compared with two yielded a 20% increase in the detection rate of CMV. For urine, tissue, and BAL specimens, inoculation of two shell vials compared with one resulted in increases of 7, 10, and 5%, respectively. For maximum detection of CMV in shell vial cell cultures, at least three vials should be inoculated with blood specimens, and two vials should be used for urine, tissue, and BAL samples.