不同方法检测淋巴瘤病理标本EB病毒的比较

目的 :探讨用不同方法检测淋巴瘤标本中EB病毒LMP 1的表达率以选择一种敏感、可靠的检测手段。方法 :运用SP法、CSA法检测EB病毒LMP 1在Raji细胞株细胞学涂片、12 3例淋巴瘤 (其中 10 4例NHL ,19例HL)切片中的表达 ,部分病例结合PCR加以验证。结果 :(1)细胞学涂片SP阳性细胞率为 70±10 % ,CSA法 10 0 %阳性。 (2 )HL中 ,SP法检测LMP 1阳性率为 6 / 19(32 % ) ,CSA法阳性率 8/ 19(4 2 % ) ,PCR阳性率为 11/ 19(5 8% )。 (3)非霍奇金淋巴瘤 (NHL)中SP法阳性率为 3/ 10 4 (其中B NHL 1/ 79,1 2 6 % ,T NHL2 / 2 5 ,8% )。CSA法阳性率为 9/ 10 4 ,其中B NHL4 / 79(5 % ) ,T NHL 5 / 2 5 (2 0 % ) ,ITCL达 2 / 10 (2 0 % )。结论 :SP法在检测低丰度抗原时漏检率较高 ,CSA法为一种较敏感、可靠的检测手段 ,结合PCR方法可有效检测低丰度抗原。     

应用激光显微切割技术检测单个结肠细胞的基因表达

目的：探讨检测单个结肠细胞的基因表达的方法。方法：应用激光显微切割技术(1aser micmdissection)从冰冻切片上将单个结肠细胞切下,提取总RNA,将RNA逆转录成cDNA,采用巢式逆转录聚合酶链反应(nested RT—PCR)检测mRNA的表达。结果：在显微镜下用紫外激光显微切割机,将单个结肠细胞成功切下,提取RNA后,逆转录成cDNA,经过巢式RT—PCR扩增后,扩增产物在琼脂糖凝胶上清晰可见。结论：联合应用激光显微切割和巢式RT—PCR可以检测单个结肠细胞的基因表达。     

沙门菌常见O抗原和H抗原荧光PCR检测方法评估

目的:评估沙门菌常见A~F血清群O抗原和H抗原荧光PCR检测方法对不同血清型沙门菌的检测效果。方法:选用不同血清型沙门菌制备模板,运用沙门菌O抗原A~F群及不同H抗原荧光PCR检测试剂盒对这些菌株进行扩增检测,分别计算不同血清型检测结果的灵敏度、特异度、检测下限、假阳性率、假阴性率、Kappa值等指标并进行分析。结果:对7种不同沙门菌O抗原的检测灵敏度为85.71%~100.00%,特异度为66.67%~100.00%;对7种不同沙门菌H抗原的检测,H1,5及Hi-l的灵敏度不高,分别为35.71%及57.14%,但特异度均较高(87.50%~100.00%);经Fisher精确检验,除H1,5阳性检测的P0.05(P=0.162),其余为P0.05;C1、C2、D1、E、F血清群及H抗原1,6、1,7、g,s,t、g,m的检测结果与血清凝集结果的一致性较高(Kappa值均大于0.75),而H1,5及Hi-l的检测结果与血清凝集结果的一致性很差(Kappa值分别为0.302及0.492);试剂盒对不同O/H抗原核酸检测扩增效率较好,最低检出限为7~137608拷贝/反应。结论:除H1,5抗原外,该荧光PCR检测试剂盒对沙门菌O/H抗原不同血清型的检测效果较好,优于传统血清凝集方法,可显著提高血清型鉴定时效,有利于疾病或暴发的早期发现。     

肿瘤患者化疗后人巨细胞病毒活动性感染检测

探讨肿瘤患者化疗后人巨细胞病毒感染检测方法的应用价值。使用免疫组化法、酶联免疫吸附试验检测IgG/M抗体,以及实时荧光定量(FQ-PCR)检测HCMV DNA。47份全血标本中抗原阳性率为48.9%,平均抗原阳性细胞数7.9±8.1(1-65)/5×104WBC,HCMV DNA阳性率19.1%(10/47),HCMV DNA含量均值为6.320×105copies,白细胞HCMV-DNA阳性率51%(25/47),HCMV DNA含量均值为3.830×107 copies,HCMV pp65抗原阳性率为48.9%(23/47),IgG抗体均阳性,IgM抗体阳性率为23.4%(12/47),以PP65抗原阳性为对照,IgM抗体检测的敏感率仅为49.3%。在连续动态检测HCMV多种指标时,结合DNA及抗原动态检测具有更高临床应用价值。     

随机扩增多态性技术联合荧光定量高敏检测三种疱疹病毒方法的建立

目的:建立一种随机扩增多态性技术(RAPD)联合荧光定量聚合酶链式反应(q PCR)高敏定量检测单纯疱疹病毒(HSV)、人类巨细胞病毒(HCMV)、水痘带状疱疹病毒(VZV)的新方法。方法:根据文献筛选出数十条随机引物,分别对三种疱疹病毒进行随机扩增,产物经2%琼脂糖凝胶电泳,选取稳定清晰条带进行分离、纯化及克隆测序,应用blast-nr比对genebank现有病毒序列,选取高于99%匹配度的基因序列作为目的片段,并在其内部用primer3.0设计特异性内引物。经过引物筛选及条件优化后建立RAPD联合q PCR检测新方法,分别检测三种疱疹病毒。结果:经过筛选,确定了HSV、HCMV、VZV扩增灵敏性及特异性最好一组引物,建立的RAPD-q PCR可分别检测出1:10~6 HSV、1:10~5 HCMV和1:10~5 VZVDNA,而单一q PCR仅能检测1:10~3 HSV、1:10~2HCMV和1:103 VZVDNA。RAPD-q PCR相比于单一q PCR灵敏性提高100-1000倍,RAPD-q PCR扩增大于1:10~5病毒DNA得到的CT值均小于22.96±0.81,与10~2 copies/μL的标准线相距远,易于区分阴性和阳性。此外,用乙型肝炎病毒及疱疹病毒互为对照未见非特异扩增,特异性好。结论:随机扩增多态性技术联合荧光定量是一种检测三种病毒高度灵敏及特异的检测方法。     

神经胶质瘤细胞ATF5表达水平对HCMV复制的影响

人巨细胞病毒(human cytomegalovirus,HCMV)在神经胶质瘤细胞中的复制水平不一,其机制尚不清楚。本研究通过下调转录激活因子5(ATF5)在神经胶质瘤细胞中的表达,检测HCMV感染神经胶质瘤细胞后病毒复制水平的变化。首先用HCMV AD169(MOI=5)分别感染U87、SY5Y及A172细胞,观察细胞形态变化,分别在24、48、72、96、120 h取各时间点上清液检测病毒滴度;Real-time PCR检测HCMV即刻早期基因(IE2)、早期基因(UL44)、晚期基因(UL99)及ATF5的表达情况;Western-blot检测病毒基因编码蛋白及ATF5表达的情况。结果显示HCMV在U87、SY5Y细胞中复制水平与病毒在A172细胞中复制水平相比,U87、SY5Y细胞组明显高于A172细胞组(P0.05),ATF5表达在U87、SY5Y细胞组与A172细胞组相比,U87、SY5Y细胞组ATF5表达明显高于A172组(P0.05);利用慢病毒介导的RNA干扰技术下调ATF5在U87、SY5Y细胞的表达,用HCMV感染细胞检测病毒基因及蛋白的表达,结果ATF5表达下调可抑制HCMV的复制(P0.05)。以上结果表明,在胶质瘤细胞中下调ATF5表达水平可以抑制HCMV的复制水平。     

单细胞PCR体系的建立及其在CRISPR/Cas9靶点活性检测中的应用

单细胞PCR是以单个细胞所含的DNA或RNA为模板进行扩增,因此其模板的制备是影响整个反应成功与否的关键因素。利用三种不同蛋白酶K细胞裂解液(SDS细胞裂解液、NP-40细胞裂解液、Tween-20细胞裂解液)分别对体外培养成熟的单个猪卵母细胞进行裂解,并直接作为模板进行PCR扩增,结果表明:NP-40细胞裂解液制备的模板质量最好,其扩增效率为95%;其次为Tween-20细胞裂解液,其扩增效率为65%;SDS细胞裂解液产物中未检测到阳性条带。结合显微注射技术对sgRNA靶点活性进行检测,结果表明利用单细胞PCR结合显微注射的方法,在受精卵内对CRISPR/Cas9靶点活性进行检测确实可行,检测结果真实可靠。     

重组人巨细胞病毒(HCMV)优势表位嵌合抗原的构建表达以及捕获法检测IgM抗体

通过计算机软件分析选择巨细胞病毒优势抗原表位, 用聚合酶链式反应(PCR)扩增优势表位基因, 构建了巨细胞病毒多优势表位嵌合抗原表达载体p4T-UL32-UL44-UL80a-UL83, 转化宿主菌大肠杆菌BL21(DE3)进行表达, GST亲和层析法获得高纯度嵌合抗原, 辣根过氧化物酶(HRP)标记嵌合抗原, 并构建捕获法检测血清中的抗HCMV IgM抗体。结果表达的优势表位嵌合抗原具有很好的抗原性, 用其建立的捕获法检测确诊的30份阳性和63份儿童阴性血, 阳性检出率和阴性检出率都是100% 说明用嵌合抗原构建的捕获法检测血清中抗HCMV IgM抗体具有更高的灵敏度和检出正确率, 其检测水平已经达到国外的同类产品水平。     

甲型肝炎病毒在人胚肺二倍体细胞核酸增殖培养模型的建立

目的采用人胚肺二倍体细胞(2BS细胞)培养,结合实时荧光定量逆转录PCR(qRT-PCR)检测,开展甲型肝炎病毒(HAV)在2BS细胞核酸增殖培养模型的建立研究。方法将不同浓度的HAV活病毒感染2BS细胞,分别于感染后0、8、10、14、20 d收获HAV感染的细胞,应用已建立的HAV核酸检测和抗原检测方法分别检测HAV病毒核酸含量和抗原含量的动态变化。结果 HAV在2BS细胞中可有效复制和扩增,高剂量组以1×10~4 CCID_(50)/mL HAV感染0 d即可检测到HAV核酸,10～20 d病毒核酸达到峰值7.607 lg copies/μL;中剂量组以1×10~2 CCID_(50)/mL HAV感染6 d可检测到HAV核酸,6～10 d病毒增殖明显(P0.05),14～20 d病毒核酸含量达到峰值7.074 lg copies/μL并趋于稳定;而低剂量组以1 CCID_(50)/mL HAV感染后,10 d HAV开始增殖,20 d病毒核酸含量与中、高剂量组别相近且基本达到峰值;当HAV感染2BS细胞10 d时,病毒核酸检出拷贝数与感染HAV浓度呈良好的线性关系,可根据标准曲线准确反映样品中的HAV活病毒含量。细胞培养结合病毒抗原表达也可有效反映HAV的扩增情况,但较病毒核酸的出现呈明显滞后,且当样品中抗原含量低时,至少需要20 d以上才可完全检测到病毒抗原的表达。结论应用2BS细胞培养和qRT-PCR检测建立了2BS细胞HAV核酸增殖模型,可快速、准确地反映HAV活病毒在细胞中的动态感染和增殖情况,可应用于HAV活病毒的快速定量、病毒特性、药物筛选和疫苗评价等相关研究。     

流行性感冒病毒细胞培养阳性相关影响因素分析

目的分析影响流行性感冒病毒(流感病毒)细胞培养阳性的相关因素。方法对2014年4月—2017年3月采集的流感样病例咽拭子标本进行PCR核酸检测,对检测阳性的样本进行细胞分离培养,以分离得到的阳性样本为病例组,阴性样本为对照组,进行对照分析研究,用Logistic回归对影响病毒分离阳性的因素进行分析。结果1 298份PCR核酸检测阳性样本中,分离阳性率为22.80%(296/1 298)。其中,季节性H3N2占13.79%(179/1 298),甲型流感病毒H1N1占1.54%(20/1 298),乙型流感病毒(Victoria)占7.24%(94/1 298),乙型流感病毒(Yamagata)占0.23%(3/1 298)。样本来源、发病采样时间间隔、原始样本PCR核酸检测结果、不同采样机构和采样至收样时间间隔对病毒分离结果差异均有统计学意义(P0.05)。结论缩短收样至接种时间间隔以及提高医疗机构的采样水平是提高流感病毒分离阳性率的关键。     

Role of Milk Whey in the Transmission of Human Cytomegalovirus Infection by Breast Milk

Breast-fed infants are susceptible to human cytomegalovirus (HCMV) infection via breast milk. In our previous study, HCMV was isolated more frequently from breast milk at later than one month after delivery than from colostrum or early breast milk. To clarify the role of milk cells and whey in vertical infection by breast feeding, we separated breast milk into milk cells and whey and examined each fraction for the presence of HCMV. We collected breast milk from mothers who breast-fed their infants (aged from 3 days to 2 months). The breast milk was centrifuged and separated into the middle layer (layer of milk whey) and the pellet (containing milk cells). We attempted to isolate HCMV from whey and to detect HCMV immediate early (IE) DNA in both milk whey and cells. HCMV was isolated from 7 out of 35 (20.0%) whey samples and HCMV IE DNA was detected from 15 out of 35 (42.9%) whey and/or milk cells. Detection rates of HCMV IE DNA in the whey layer and milk cells were 39.1% (25 out of 64) and 17.2% (11 out of 64), respectively. HCMV IE DNA was not detected in colostrum, but was detected in breast milk samples one month after delivery. Therefore, cell-free HCMV shed into milk whey may have a more important role in vertical infection by breast milk than cell-associated HCMV in the milk.     

Dominance of virus over host factors in cross-species activation of human cytomegalovirus early gene expression

Human cytomegalovirus (HCMV) exhibits a highly restricted host range. In this study, we sought to examine the relative significance of host and viral factors in activating early gene expression of the HCMV UL54 (DNA polymerase) promoter in murine cells. Appropriate activation of the UL54 promoter at early times is essential for viral DNA replication. To study how the HCMV UL54 promoter is activated in murine cells, a transgenesis system based on yeast artificial chromosomes (YACs) was established for HCMV. A 178-kb YAC, containing a subgenomic fragment of HCMV encompassing the majority of the unique long (UL) region, was constructed by homologous recombination in yeast. This HCMV YAC backbone is defective for viral growth and lacks the major immediate-early (IE) gene region, thus permitting the analysis of essential cis-acting sequences when complemented in trans. To quantitatively measure the level of gene expression, we generated HCMV YACs containing a luciferase reporter gene inserted downstream of either the UL54 promoter or, as a control for late gene expression, the UL86 promoter, which directs expression of the major capsid protein. To determine the early gene activation pathway, point mutations were introduced into the inverted repeat 1 (IR1) element of the UL54 promoter of the HCMV YAC. In the transgenesis experiments, HCMV YACs and derivatives generated in yeast were introduced into NIH 3T3 murine cells by polyethylene glycol-mediated fusion. We found that infection of YAC, but not plasmid, transgenic lines with HCMV was sufficient to fully recapitulate the UL54 expression program at early times of infection, indicating the importance of remote regulatory elements in influencing regulation of the UL54 promoter. Moreover, YACs containing a mutant IR1 in the UL54 promoter led to reduced ( approximately 30-fold) reporter gene expression levels, indicating that HCMV major IE gene activation of the UL54 promoter is fully permissive in murine cells. In comparison with HCMV, infection of YAC transgenic NIH 3T3 lines with murine cytomegalovirus (MCMV) resulted in lower (more than one order of magnitude) efficiency in activating UL54 early gene expression. MCMV is therefore not able to fully activate HCMV early gene expression, indicating the significance of virus over host determinants in the cross-species activation of key early gene promoters. Finally, these studies show that YAC transgenesis can be a useful tool in functional analysis of viral proteins and control of gene expression for large viral genomes.     

The 21bp repeat element of the human cytomegalovirus major immediate early enhancer is a negative regulator of gene expression in undifferentiated cells.

The major immediate early regulatory region of human cytomegalovirus (HCMV) has a complex set of DNA sites through which both cellular and viral factors coordinately regulate immediate early gene expression. In undifferentiated human teratocarcinoma (T2) cells we have previously shown that major immediate early gene expression is repressed by a differentiation specific nuclear factor MBF1, which binds to the imperfect dyad symmetry located upstream of the enhancer. However, upon differentiation MBF1 decreases resulting in immediate early gene expression. In this study we show, by mobility shift analysis that the same or similar factor(s) also binds to the 21bp repeat of the major immediate early enhancer. Deletion of this 21bp repeat from the immediate early enhancer expression vectors results in increased CAT expression in undifferentiated T2 cells, to levels similar to that in differentiated cells. Consequently, the 21bp repeat of the HCMV enhancer also acts to negatively regulate major immediate early enhancer function in non-permissive cells.     

An improved immunocytochemical method for the detection of human cytomegalovirus antigens in peripheral blood leucocytes

A recently described immunoperoxidase method for the detection of nuclear human cytomegalovirus (HCMV) immediate early antigen (IEA) directly on peripheral blood leucocytes suffers from the drawback that the antigen is vulnerable to endogenous peroxidase inactivation procedures. To solve this problem a procedure is developed in which endogenous peroxidase is inactivated after binding and immobilization of the primary antibody with 4% formaldehyde. In combination with this procedure, three types of inactivation were investigated: glucose/glucose oxidase, hydrochloric acid and methanol/H2O2. Of these three, the first gives optimal results, especially in combination with methanol/acetic acid (20/1 v/v) as the primary fixative. This procedure results in preparations which allow for a more objective evaluation and enable automated examination using bright field microscopy. As a second improvement we developed a simple adherence method in order to diminish the risk of infection for the laboratory staff during processing of unknown blood samples. The protocol described shows great clinical potential for the diagnosis of HCMV infections.     

肾移植术后巨细胞病毒pp65检测的临床意义

目的探讨肾移植受者术后,巨细胞病毒被膜磷蛋白pp65的检测在活动性巨细胞病毒感染中的意义。方法用间接免疫荧光法检测肾移植受者术后HCMV-pp65,同时用酶联免疫捕获法检测HCMV-IgM抗体,共采集91份血标本。结果 91份血标本中,HCMV-pp65阳性24份（26.4%）,HCMV-IgM抗体阳性4份（4.4%）,在24例HC-MV-pp65抗原血症阳性的患者中,有20例出现HCMV感染症状及HCMV病。结论 HCMV-pp65在活动性巨细胞病毒感染的检测中具有早期、准确的优点,可辅助临床对HCMV感染进行早期诊断与治疗。     

Expression of human cytomegalovirus immediate early antigens is responsible for a novel chromatin structure of infected human embryonic lung cells

Whereas human embryonic lung (HEL) cells displayed chromatin fibers composed of a repeat of conventional nucleosomes of 15 nm in diameter, human cytomegalovirus (HCMV) infection induced transient appearance of a novel chromatin structure composed of a repeat of large ellipsoids of 45-65 nm X 15-30 nm with linkers of 50-60 nm long and 6-7 nm thick. Essentially the same change in chromatin structure could be induced when uninfected HEL cell nuclei were incubated in vitro with a 0.4 M NaCl nuclear extract from HCMV-infected HEL cells expressing immediate early antigens (IEA's) or with a similar nuclear extract from NIH/3T3 cells constitutively expressing HCMV IEA's. The latter cell line was established by transformation of the mouse cells with a plasmid carrying the HCMV major immediate early and immediate early 2 genes. These results together with those of control experiments suggest that the expression of IEA's is directly or indirectly responsible for the appearance of the novel chromatin structure in HCMV-infected HEL cells.     

Human cytomegalovirus immediate early proteins and cell growth control

It is widely accepted that small DNA tumor viruses, such as adenovirus, simian virus 40 and papillomavirus, push infected cells into S-phase to facilitate the replication of their genome. Until recently, it was believed that the large DNA viruses (i.e. herpesviruses) functioned very differently in this regard by inducing a G₁ arrest in infected cells as part of their replication process. However, studies over the last 6–8 years have uncovered striking parallels (and differences) between the functions of the major immediate early (IE) proteins of at least one herpesvirus, human cytomegalovirus (HCMV) and IE equivalents encoded by small DNA tumor viruses, such as adenovirus. Similarities between the HCMV major IE proteins and adenovirus IE proteins include targeting of members of the RB and p53 families and an ability of these viral factors to induce S-phase in quiescent cells. However, unlike the small DNA tumor virus proteins, individual HCMV IE proteins target different RB family members. HCMV also encodes several other IE gene products as well as virion tegument proteins that act early during infection to prevent an infected cell from replicating its host genome and from undergoing apoptosis. Here, we review the specifics of several HCMV IE proteins, two virion components, and their functions in relation to cell growth control.