应用PCR法检测新生儿肝炎综合征患者HCMV-DNA

应用PCR检测30例临床诊断为新生儿肝炎综合征（NHS）患者和14例正常新生儿尿中人巨细胞病毒DNA（HCMV－DNA）。结果阳性率分别为66．7％和14．29％，两者差异显著。HCMV可能为NHS的主要病原．同时对30例NHS患儿尿进行HCMV分离，阳性率为53．3％。PCR与病毒分离的符合率为100％，PCR方法快速、简便、灵敏、安全、特异，适用于临床诊断和研究．     

应用地高辛标记HCMV DNA混合探针检测人巨细胞病毒DNA

本文比较了~(32)P和地高辛标记的HCMV EcoRI单一Z片段与EcoR IZ、K、J混合片段探针的敏感性。结果~(32)P和地高辛分别标记的EcoRIZ单一片段检测敏感性分别为50pg和1pg,而两者标记的EcoRI Z、K、J混合片段检测敏感性分别提高到5pg和0.1pg。地高辛标记的混合片段探针比~(32)P标记单一EcoRIZ片段探针敏感性提高500倍,比~(32)P标记混合探针高50倍,比地高辛标记的EcoRI Z片段探针高10倍。应用地高辛标记混合片段探针检测了正常小儿尿标本10份,阳性率为60％(6/10),与免疫组化方法对比,两者符合率为100％。检测肾移植患者尿标本33份,阳性率为46％(15/33);白血病患者尿标本18份,阳性率为72％(13/18),与PCR方法对比两者符合率为93％。地高辛标记混合探针可快速、敏感、特异地检测人巨细胞病毒感染。     

人呼吸道合胞病毒荧光PCR检测方法的建立和评价

目的建立人呼吸道合胞病毒(hRSV)荧光PCR(FQ-PCR)检测方法,并确定其最低检出限。方法针对hRSVN基因相对高度保守的序列,采用引物和探针设计软件PrimerExpressv2.0,设计1对特异性引物和1条TaqMan荧光探针,组装成荧光PCR检测试剂。以此试剂检测345份咽拭子样品,与ELISA法检测hRSVIgM抗体比较,确定最低检出限。结果该方法的最低检出限是传统的病毒滴度测定的104.79倍。与IgM抗体检测法相比,对于感染早期患者具有更高的检出率。结论建立的hRSVFQ-PCR检测方法具有简便、快捷的特点,适用于hRSV感染的早期诊断。     

巨细胞病毒感染PCR检测方法的建立

目的建立检测巨细胞病毒污染的PCR检测方法,并制备检测试剂盒。方法采用TaqMan探针,选择保守序列CMV-pp65设计引物,荧光定量病毒载量;对试剂盒进行敏感性、重复性和特异性等测试;对31份临床阳性血清标本和106份正常献血者血浆标本进行检测,并与国内同类试剂盒进行比较。结果该试剂盒检测线性定量范围可达202~3.6×109copies/ml,敏感性为360copies/ml,CV值在1%左右,特异性高,临床标本检测的符合率为100%。本试剂盒与国内已上市试剂盒比较,具有较好的相关性、更高的敏感性和更宽的定量检测范围。结论制备的试剂盒特异性和敏感性均较高,适用于临床上的病毒检测。     

人巨细胞病毒基因克隆及分子探针的临床应用

以 PuC18为载体克隆了人巨细胞病毒(HCMV)AD169株 ECoRI 部分基因组片段。以α~(-32)P 标记的 ECoRI-Z 片段为探针,对尿、脐带血和宫颈分泌物等标本进行了 HCMV DNA 的临床诊断分析。该探针可以检测出12pg 水平的 HCMV DNA,但与人单纯疱疹病毒Ⅰ型(HSVI)和人单纯疱疹病毒Ⅱ型(HSVⅡ)、λ噬菌体、Puc18载体 DNA 等不杂交。以斑点杂交法检测了正常新生儿尿标本50份和临床诊断为新生儿黄疸的尿标本109份,其阳性率分别为8%和36%;此外还检测了172份随机收集的新生儿脐带血白细胞 DNA,其阳性率为15%,说明了我国新生儿 HCMV 感染的严重性。检测结果亦证实患宫颈炎和宫颈癌的患者,HCMV DNA 检出率明显高于正常妇女,说明分子杂交法可用于各种临床标本 HCMV DNA 的检测。     

人巨细胞病毒ISCOMs的研制

应用巨细胞病毒被膜蛋白与Quil A结合制备ISCOMs疫苗,经小鼠的免疫学实验发现,ISCOMs实验组IgG及IgM水平明显高于其它对照组,其IgG1、IgG2a、IgG2b也较其它组为高。提示该疫苗有可能在预防人体HCMV原发感染中发挥作用。     

人巨细胞病毒融合表达载体的构建及鉴定

目的构建含人巨细胞病毒(HCMV)gB680糖蛋白基因和突变的pp65蛋白基因(pp65m)的融合表达载体,并检测其在COS-7细胞中的表达。方法用PCR法从HCMV基因组中钓取gB680和pp65m蛋白基因,分别亚克隆入pMD18-T载体中,经酶切鉴定并测序后,构建真核表达载体pVAX1/gB680+pp65m,以ELISA法检测其在COS-7细胞中的瞬时表达。结果重组质粒含有gB680和pp65m融合基因,并能在COS-7细胞中表达。结论已成功构建了人巨细胞病毒融合表达载体,并可在真核细胞中表达。     

人巨细胞病毒疫苗的研究现状及存在的问题

人巨细胞病毒(HCMV)在人群中的感染非常普遍,所造成的后果也很严重。HCMV是导致免疫抑制或免疫缺陷患者发病率和死亡率高的重要原因。孕妇原发或继发HCMV感染均可引起新生儿宫内感染或围产期感染,是目前引起胎儿出生缺陷的主要原因。因此,研制一种可用于保护这些高危人群的HCMV疫苗,具有重大的公共卫生学意义。虽然到目前为止,还没有一种HCMV疫苗获准上市,但是对一些关键的病毒靶蛋白的研究已经取得了重要的成果。最佳的疫苗方案还有待于进一步的临床观察。本文就HCMV疫苗研究的最新进展、存在的主要问题、前景等进行综述。     

人巨细胞病毒ISCOMs对小鼠细胞免疫应答的调节作用

应用人巨细胞病毒被膜蛋白制备实验型免疫刺激复活物 (ISCOMs)疫苗能诱发细胞免疫反应。对小鼠 1次接种ISCOMs未能诱生明显细胞毒性T细胞 (Tc)及自然杀伤细胞 (NK)细胞活性增加 ,但能使L3T4及其与LyT2的比值增加。第 2次免疫后小鼠Tc及NK细胞活性明显增加。提示该疫苗有可能在预防人巨细胞病毒 (HC MV)原发感染中发挥作用。     

人巨细胞病毒培养条件的优化及快速病毒滴度检测方法的建立

目的优化人巨细胞病毒(human cytomegalovirus,HCMV)的培养条件,并建立一种快速检测HCMV滴度的方法。方法以人胚肺二倍体细胞(MRC-5)为病毒传代和滴度测定用细胞,对病毒接种量、培养温度、保护剂种类等条件进行优化。将本实验室建立的快速微量中和试验-感染细胞核染色法用于病毒滴度检测,并与蚀斑形成法及微量细胞病变法进行比较,采用Behrens-Karher法计算病毒滴度。结果 HCMV接种MRC-5细胞后在37℃培养可出现明显病变;MOI为1.0时,病毒滴度基本达到平台;病毒收获时加入1%人血白蛋白或10%FBS保护剂可维持病毒滴度稳定。同一病毒液经蚀斑形成法检测病毒滴度为5.707 lg PFU/ml,微量细胞病变法和感染细胞核染色法检测病毒滴度分别为5.633和5.667 lg CCID50/ml,3种方法的检测结果差异无统计学意义(P0.05)。结论HCMV接种MRC-5的最佳培养条件为:病毒接种量1.0 MOI,在37℃培养,加入1%人血白蛋白或10%FBS作为保护剂。建立的感染细胞核染色法可用于HCMV滴度的快速测定,为抗病毒物质的开发及效果评价提供了一种新的技术手段。     

菌落PCR技术检测化妆品中金黄色葡萄球菌

利用菌落PCR技术,以金黄色葡萄球菌的特有基因femA为靶基因,选择特异引物．进行扩增,鉴定化妆品中金黄色葡萄球菌。琼脂糖凝胶电泳检测PCR产物结果显示,仅在含有金黄色葡萄球菌基因组的样品中得到条带。     

Detection of Mycoplasma Contamination in Transplanted Retinal Cells by Rapid and Sensitive Polymerase Chain Reaction Test

Contamination of cells/tissues by infectious pathogens (e.g., fungi, viruses, or bacteria, including mycoplasma) is a major problem in cell-based transplantation. In this study, we tested a polymerase chain reaction (PCR) method to provide rapid, simple, and sensitive detection of mycoplasma contamination in laboratory cultures for clinical use. This mycoplasma PCR system covers the Mycoplasma species (spp.) listed for testing in the 17th revision of the Japanese Pharmacopoeia, and we designed it for use in transplantable retinal cells. Here, we analyzed mycoplasma contamination in induced pluripotent stem cell (iPS cell)-derived transplantable retinal pigment epithelium (RPE) cells. In the spike tests to RPE cells with nine species of class Mollicutes bacteria, including seven Mycoplasma spp. and one of each Acholeplasma spp. and Ureaplasma spp., contamination at the concentration of 100 and 10 CFU/mL were detected with 100% probability in all cases, while 1 CFU/mL had a detection rate of 0–75%. DNA prepared from bacteria species other than class Mollicutes species was not detectable, indicating the specificity of this PCR. While iPS cells and iPS-RPE cells established in our laboratory were all negative by this PCR, some of the commercially available cell lines were positive. Cells for transplantation should never have infection, as once pathogens are implanted into the eyes, they can cause severe intraocular inflammation. Thus, it is imperative to monitor for infections in the transplants, although generally, mycoplasma infection is difficult to detect.     

用于基因扩增热循环温度跟踪的前馈变结构PID控制技术

To track the rapidly changing temperature profiles of thermal cycling of polymerase chain reaction （PCR） accurately, an innovative feedforward variable structural proportional-integral-derivative （FVSPID） controller was developed. Based on the step response test data of the heat block, a reduced first order model was estabfished at different operating points. Based on the reduced model, the FVSPID controller combined a feedforward path with the variable structural proportional-integral-derivative （PID） control. The modified feedforward action provided directly the optimal predictive power for the desired setpoint to speed up the dynamic response. To cooperate with the feedforward action, a variable structural PID was applied, where the P mode was used in the case of the largest errors to speed up response, whereas the PD mode was used in the case of larger errors to suppress overshoot, and finally the PID mode was applied for small error conditions to eliminate the steady state offset. Experimental results illustrated that compared to the conventional PID controller, the FVSPID controller can not only reduce the time taken to complete a standard PCR protocol, but also improve the accuracy of gene amplification.     

3′ Tth Endonuclease Cleavage Polymerase Chain Reaction (3TEC-PCR) Technology for Single-Base-Specific Multiplex Pathogen Detection using a Two-Oligonucleotide System

Polymerase chain reaction (PCR) is the standard in nucleic acid amplification technology for infectious disease pathogen detection and has been the primary diagnostic tool employed during the global COVID-19 pandemic. Various PCR technology adaptations, typically using two-oligonucleotide dye-binding methods or three-oligonucleotide hydrolysis probe systems, enable real-time multiplex target detection or single-base specificity for the identification of single-nucleotide polymorphisms (SNPs). A small number of two-oligonucleotide PCR systems facilitating both multiplex detection and SNP identification have been reported; however, these methods often have limitations in terms of target specificity, production of variable or false-positive results, and the requirement for extensive optimisation or post-amplification analysis. This study introduces 3′ Tth endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, Tth endonuclease IV, for real-time multiplex detection and SNP identification. Complete analytical specificity, low limits of detection, single-base specificity, and simultaneous multiple target detection have been demonstrated in this study using 3TEC-PCR to identify bacterial meningitis associated pathogens. This is the first report of a two-oligonucleotide, real-time multiplex PCR technology with single-base specificity using Tth endonuclease IV.     

Efficiency of the Polymerase Chain Reaction

The polymerase chain reaction (PCR) has found wide application in biochemistry and molecular biology such as gene expression studies, mutation detection, forensic analysis and pathogen detection. Increasingly quantitative real time PCR is used to assess copy numbers from overall yield. In this study the yield is analyzed as a function of several processes: (1) thermal damage of the template and polymerase occurs during the denaturing step, (2) competition exists between primers and templates to either anneal or form dsDNA, (3) polymerase binding to annealed products (primer/ssDNA) to form ternary complexes and (4) extension of ternary complexes. Explicit expressions are provided for the efficiency of each process, therefore reaction conditions can be directly linked to the overall yield. Examples are provided where different processes play the yield-limiting role. The analysis will give researchers a unique understanding of the factors that control the reaction and will aid in the interpretation of experimental results.     

间苯二胺扩链剂酰基化合成聚脲反应动力学研究

采用酰基化方法对扩链剂间苯二胺进行了改性,以减缓聚脲的高反应活性来研究合成反应的动力学方程。以间苯二胺、冰乙酸为原料,磷酸为催化剂合成了新型位阻型扩链剂二乙酰间苯二胺,并用红外光谱、高分辨质谱、凯氏定氮法和核磁共振氢谱分析确定了合成产物的化学结构。将它和端氨基聚醚、4,4’-二苯甲烷二异氰酸酯(MDI)通过两步溶液法合成了新型聚脲,考察了改性二胺扩链剂对聚脲性能的影响,并用傅里叶变换红外光谱仪(FTIR)跟踪该固化反应过程,得到了不同温度下的反应速度常数。实验结果表明,当用改性扩链剂代替原扩链剂合成聚脲时,凝胶时间大大延长。该反应体系表现为良好的一级动力学关系,并得出该反应的活化能。     

Label-Free in Situ Monitoring of the DNA Hybridization Chain Reaction by Using Sequence-Selective Minor-Groove-Binding Fluorophores

A new label-free in situ monitoring system for the hybridization chain reaction (HCR) based on DNA minor-groove-binding fluorophores [Hoechst 33258 (Hoe) or quinone cyanine-dithiazole (QCy-DT)] has been developed. Use of two unmodified hairpin oligodeoxyribonucleotides containing incomplete double-stranded AATT sequences enabled target-dependent formation of probe binding sites—that is, AATT double strand—in the HCR product, together with fluorescence enhancement of minor-groove-binding fluorophores in situ. This system allows target DNA to be detected through the fluorescence enhancement of Hoe and QCy-DT in real time and in situ. Further development of a label-free, isothermal detection system might provide a cost-effective and user-friendly method for nucleic acid detection.     

人T淋巴细胞CD3ε链的原核表达及纯化

目的原核表达并纯化人T淋巴细胞CD3ε(hCD3ε)链。方法以健康成人外周血单个核细胞总RNA为模板,采用RT-PCR法扩增人CD3ε链基因,并克隆入pCR-II载体,酶切鉴定及测序分析后,再定向克隆至原核表达载体pGEX-4T-3中,构建重组表达质粒pGEX-4T-3-hCD3ε,转化E.coli BL21(DE3),IPTG诱导表达。表达产物经GST亲和层析纯化后,进行Western blot鉴定。结果酶切分析及DNA测序证实,hCD3ε链基因被正确克隆入pGEX-4T-3载体,并能在原核系统中稳定表达。表达产物的相对分子质量为49 500,0.2 mmol/L IPTG 25℃诱导表达4.5 h,目的蛋白的表达量最高,占菌体总蛋白的29.3%,其中可溶性表达占12.8%。纯化的融合蛋白纯度可达86.1%,可分别被兔抗人CD3ε多抗和羊抗GST多抗识别。结论已成功地原核表达并纯化了GST-hCD3ε融合蛋白。