免疫—PCR技术进展

免疫－ＰＣＲ结合了抗原抗体反应的特异性和ＰＣＲ的高敏感性,是一种极为敏感的抗原检测技术,并适合于各种微量抗原的检测。免疫－ＰＣＲ的关键在于形成抗体－标记ＤＮＡ偶联物,作为桥梁连接免疫反应的特异和ＰＣＲ的高扩增能力。本文简述了－ＰＣＲ的基流程以及与标ＤＮＡ相偶联,ＰＣＲ产物检测方法的进展。     

免疫—PCR：一种新的抗原检测系统

免疫－ＰＣＲ是１９９２年建立的抗原检测系统，其基本过程和ＥＬＩＳＡ相似，免疫－ＰＣＲ用一段可扩增的ＤＮＡ分子代替ＥＬＩＳＡ中的酶来放大检出信号，因而大大提高了灵敏度，尽管此法尚处于探索阶段，但应用前景是诱人的。     

一种克隆PCR产物的新方法

一种克隆ＰＣＲ产物的新方法周复春,李学伍,吴斌,陈焕春（华中农业大学牧医系,武昌）基因克隆和分离是分子生物学和细胞生物学研究必不可少的组成部分。ＰＣＲ基因扩增可以产生微克级的特异性刀ＮＡ片段,可以简化从基因组ＤＮＡ克隆单拷贝基因片段的步骤。到达这种数...     

一种简易的免疫PCR方法的建立

免疫PCR为一种高敏感度检测抗原的新技术,操作程序大多沿袭ELISA方法.用戊二醛作连接剂,将蛋白质高效率包被在普通PCR管内壁,使免疫及PCR反应用普通PCR仪得以在管中连贯地进行.实验结果表明,标准曲线的线性关系好,与ELISA方法比较敏感度高出约10⁵.这一改良法的建立,可望促进免疫PCR的普及应用.     

原位PCR技术及其应用

原位ＰＣＲ技术是通过在染色体上或组织细胞内对靶核酸序列进行原位扩增,用原位杂交技术检测,从而对靶核酸进行定性,定位和定量分析的研究方法,原位ＰＣＲ大大提高了原位杂交技术的灵敏度,在分子生物学基础理论和临床医学研究方面展示了美好的应用前景。     

PCR技术的应用与微生物系统进化研究的发展

微生物的分类进化研究是生命学科的基础理论研究。近年来随着生物技术的日趋完善,以及分子生物学的不断渗透,特别是聚合酶链反应技术的出现,使人们将系统进化的研究从宏观逐渐转向微观,以便更准确地反映生物体间真正的进化关系。     

免疫——PCR：继续还是放弃？

从刚刚建立的一种新的检测方法出发，对免疫－ＰＣＲ的原理及其应用前景作了一番探讨，并与其它类似的检测方法进行了比较，指出了它的特点、优越性和不足之处。针对免疫－ＰＣＲ的缺点提出了一些改进的方案，以使其朝着实际应用方面迈进一步。     

Immuno-PCR法诊断早期梅毒的方法学建立及其应用

目的建立Immuno-PCR法诊断早期梅毒的方法学,评价其灵敏度、特异性、重复性及其临床应用。方法利用基因重组TpN47抗原免疫新西兰兔,制备抗体并用Weston blotting检测;利用抗TpN47抗体作为捕获抗体与血清中TpN47抗原结合,通过链霉亲和素、生物素化抗体、生物素化DNA和PCR扩增等建立Immuno-PCR法检测梅毒螺旋体抗原TpN47体系;评价该方法的灵敏度、特异性和重复性;收集200例临床标本通过Immuno-PCR法、ELISA、TPPA和TURST法进行临床应用比较。结果 Weston blotting结果显示TpN47抗体阳性;Immuno-PCR比ELISA法敏感性强103倍,比TPPA、TURST强105倍;特异性高,重复性好。临床标本中Immuno-PCR法敏感性和特异性分别为86.00%（P〈0.05）和100.00%,ELISA法为71.00%和98.00%,TPPA法为65.00%和100.00%,TRUST法为68.00%和95.00%。结论 Immuno-PCR法检测梅毒螺旋体TpN47抗原敏感性高,特异性强,重复性好,可作为梅毒螺旋体感染的早期诊断方法 。     

蛋白质检测新技术——邻位连接技术及初步应用

邻位连接技术（proximity ligation assay,PLA）,是新研发的一项高灵敏度的蛋白质体外分析技术。该方法利用一对邻位探针（proximity probes）对靶分子进行双识别,通过连接反应产生可扩增的检测信号,以实时 PCR进行放大和检测,将对蛋白质的检测转变成为对DNA的检测,实现痕量蛋白的分析,具有极高的检测灵敏度和特异性。综述了邻位连接技术的原理、研究进展以及该技术在蛋白质分析及疾病诊断领域的初步应用。     

Characterization of a Cloned pR72H Probe for Vibrio parahaemolyticus Detection and Development of a Nonisotopic Colony Hybridization Assay

Vibrio parahaemolyticus is a halophilic bacterium often found in shellfish and is an important causative agent of food poisoning in Taiwan. A rapid and efficient detection method is required to identify this foodborne pathogen. A 0.76-Kb HindIII DNA fragment was cloned from the chromosomal DNA of V. parahaemolyticus strain no. 93, designated as pR72H fragment, was used as a polynucleotide probe. It was labeled with digoxigenin-11-dUTP (DIG) by the random primer-labeling method. The sensitivity and specificity of the digoxigenin-labeled 0.76-Kb DNA probe was determined by colony hybridization assay. Under stringent hybridization conditions, 122 of 124 isolates of V. parahaemolyticus showed positive hybridization reaction with DIG-0.76-Kb DNA probe; the negative strains were attributed to slow growth. The DIG-0.76-Kb probe did not hybridize with 86 isolates of other vibrios and a number of other enterics as well as nonenteric microorganisms. The sensitivity and specificity of this DIG probe are 98% and 100%, respectively. This nonisotopic colony hybridization assay can be very useful for routine monitoring of V. parahaemolyticus in the food industry, environmental analysis and clinical laboratories.     

直接酶标——化学发光分子杂交体系的建立

本文通过调整发光剂、氧化剂、发光增强剂的浓度,并加入一组辅助剂,改进了依赖过氧化物酶化学发光液的配方。并通过比较实验证明:该发光液的检测灵敏度高于其它原有的发光液,同时还发现合成后的酶标复合物可经CM-32离子交换柱阶段洗脱纯化,以提高杂交灵敏度。从而建立了一套直接酶标DNA探针——化学发光自显影分子杂交体系。其狭缝杂交灵敏度可达0.03pg。并利用该体系进行了单拷贝基因分析,Western印迹杂交及检测血清HBV DNA等的应用研究。     

人博卡病毒TaqMan探针实时定量PCR检测方法的建立及初步应用

目的:建立人博卡病毒(HBoV)核酸特异、快速、敏感的TaqMan探针实时定量PCR检测方法,并对临床样本进行检测。方法:比对编码HBoV非结构蛋白NP-1的基因序列,选取其保守片段设计引物和探针,建立实时荧光定量PCR检测方法,并与传统PCR方法进行比较,然后分别对两者的灵敏性、特异性、稳定性及临床样本检验的适用性等进行评价。结果:所建立的实时定量PCR检测方法可用于HBoV的特异性检测;相对于传统PCR所达到的250拷贝/反应的检测灵敏度,实时定量PCR的检测灵敏度可高达10拷贝/反应,检测范围为109～101拷贝/反应,且具有良好的特异性和重复性;初步用于76份临床呼吸道标本检测,检出阳性5例,高于普通PCR方法(3/76)。结论:建立了HBoV TaqMan探针实时定量PCR检测方法,并可用于临床鼻咽拭子样本的检测,为开展HBoV流行病学监测及早期临床诊断提供了技术手段。     

丙型肝炎病毒免疫电镜的初步研究

采用组织匀浆免疫沉淀后负染、免疫组化块染后包埋、原位包埋等免疫电镜技术,研究丙型肝炎病毒（ＨＣＶ）。组织匀浆、免疫沉定、负染后在电镜下观察到与ＨＣＶ相关的类病毒颗粒,形态与披膜病毒相似,大小多在５５～６５ｎｍ,圆形,有包膜,边缘略有突起或比较平滑,有胶体金结合在此种颗粒上及其周围。无关单抗阴性对照无类似颗粒及胶体金。免疫酶染电镜下还见到成堆可疑颗粒。此外,ＨＣＶ－Ｅ区抗原染色后原位包埋,尚发现胶体金大多结合于大小５０ｎｍ左右圆形结构的内部,表明Ｅ区单抗针对的特异性抗原位点位于这种结构的内侧。     

New fluorescent probes for ligand-binding assays of odorant-binding proteins

Fluorescence-linked binding assays allow determination of dissociation constants at equilibrium and have recently become increasingly popular, thanks to their ease of operation. Currently used probes, such as 1-aminoanthracene and N-phenyl-1-naphthylamine, are excited and emit in the ultraviolet region, but alternative ligands operating in the visible spectrum would be highly desirable for applications in biosensing devices. Based on the two above structures, we have designed and synthesised six new fluorescent probes to be used in ligand-binding assays. The compounds are derivatives of naphatalene, anthracene and fluoranthene and present two aromatic moieties linked by an amine nitrogen. We have measured the emission spectra of the new probes and their binding to three odorant-binding proteins. The probes bind the tested proteins with different affinities, generally with dissociation constants about one order of magnitude lower than the parent compounds. The extended aromatic systems present in the new compounds produced a shift of both excitation and emission peaks at higher wavelength, close or within the visible spectrum, thus facilitating measurements in biosensors for odorants and small organic molecules using optical devices.     

Assessment of small ligand-protein interactions by electrophoretic mobility shift assay using DNA-modified ligand as a sensing probe

The interaction between a small ligand and a protein were assessed by electrophoretic mobility shift assay. A sensing probe was created by modifying the model ligand, biotin, with DNA. The complex of DNA-modified ligand and anti-biotin antibody or streptavidin as a target protein was analyzed by agarose gel electrophoresis. The band corresponding to the DNA-modified ligand was shifted in the presence of the target protein, and the intensities of the shifted bands were decreased by adding increasing concentrations of free ligand ranging from 0.1 μM to 100 μM. From this calibration the concentration of ligand in the samples could be determined, allowing for evaluation of the interaction between a small ligand and its target.     

藻胆蛋白荧光探针及其标记

藻胆蛋白是一系列新型的荧光标记探针,具有优良的荧光特性,以藻胆蛋白荧光探针标记抗体还可用于血清可溶性抗原（或抗体）的荧光免疫检测,其标记方法可分为直接法和间接法。结合藻胆蛋白的特点,研究藻胆蛋白的标记方法有助于提高荧光免疫检测的灵敏度。