诺如病毒常见流行株胶体金免疫层析快速检测方法

【背景】诺如病毒是全球引发急性胃肠炎的最主要病原之一,具有丰富的遗传多样性。【目的】建立一种简便快捷、适用于诺如病毒常见流行株的胶体金免疫层析检测方法。【方法】将抗诺如病毒衣壳蛋白的单克隆抗体1B10作为金标抗体、抗诺如病毒衣壳蛋白的单克隆抗体1D6作为检测线、羊抗鼠抗体作为质控线组装成胶体金试纸条。对试纸条的组装条件进行优化,确定最佳标记pH、金标抗体最佳浓缩比例及检测线(test line,T线)、质控线(control line,C线)最佳划线浓度等。对新方法进行性能评价,包括灵敏性试验、特异性试验、保存期试验及符合率实验等。【结果】所建立的诺如病毒胶体金试纸条检测方法最低检测浓度为5.9×105copies/μL。本方法与常见的腹泻病毒,如轮状病毒、星状病毒、腺病毒、肠道病毒均无交叉反应。批次间与批次内重复较好,保存期试验表明试纸条至少可以室温密封干燥保存一年时间。应用所建立的胶体金检测方法对24份临床粪便样本进行检测,检测结果与实时荧光RT-PCR方法的阳性符合率约为83%(15/18),常见流行株GII.2型、GII.4型、GII.17型均被成功检出。【结论】建立的胶体金试纸条检测方法具有较好的特异性与稳定性,可用于诺如病毒常见流行株检测及大规模流行病学调查。     

基于胶体金的8-羟基-2′-脱氧鸟苷免疫层析试纸条北大核心CSCD

8-羟基-2′-脱氧鸟苷(8-hydroxy-2′-deoxyguanosine,8-OHdG)是评价DNA氧化损伤较灵敏和稳定的生物标志物。文中采用竞争法建立一种快速、灵敏检测8-OHdG的胶体金免疫层析试纸条。将样品垫(玻璃纤维素膜)、结合垫(玻璃纤维素膜)、硝酸纤维素膜和吸水垫依此粘贴在聚氯乙烯(polyvinyl chloride,PVC)底板上,构建试纸条。通过柠檬酸钠还原三水合四氯金酸制备胶体金(gold nanoparticles,AuNPs),8-OHdG配对的抗体(antibody,Ab)包被于AuNPs的外层(Ab coated AuNPs,Ab@AuNPs)作为探针。牛血清蛋白(bovine serum protein,BSA)与8-OHdG用碳二亚胺盐酸盐偶联制备人工抗原,作为检测线的包被抗原。羊抗鼠多抗(imunoglobulin G,IgG)作为质控线的包被抗体。对试纸条的硝酸纤维素膜、上样液的配方、金标抗体喷涂量等实验参数进行了优化。结果表明,硝酸纤维素膜(nitrocellulose film,NC)膜采用CN 95,上样液的最优配方为1%BSA+3%吐温-20+3%蔗糖+0.9%NaCl溶液,最适金标抗体喷涂量为4μL。利用试纸条在可见光下检测8-OHdG,根据检测线(test line,T线)和质控线(control line,C线)的显色强度对比,可初步判断尿液中8-OHdG的含量水平。并通过T线的灰度值计算尿液中的8-OHdG的浓度,检测限为2.55μg/L。该方法简单、快速且有较好的特异性,可检测人体尿液中的8-OHdG含量,以初步评价人体的健康状态。     

狂犬病病毒抗体胶体金检测试纸的制备

通过胶体金免疫层析技术建立一种特异、便捷、快速的狂犬病病毒抗体检测方法,对犬等动物免疫狂犬病疫苗后的抗体水平监测提供参考。用醋酸锌沉淀法沉淀狂犬病病毒CVS11,Sepharose 4FF进行层析纯化。用柠檬酸三钠还原法制备的胶体金,标记纯化的狂犬病病毒,喷于试纸的结合释放垫 (金标垫),将SPA (葡萄球菌表面A蛋白) 和纯化的兔抗狂犬病病毒IgG分别喷于试纸的T (检测线) 处和C (对照线) 处,组装试纸条。用制备的试纸条对261份犬血清进行检测,与快速荧光灶抑制试验 (RFFIT) 检测的结果一致;对已知效价的犬狂犬病毒中和抗体 (VNA) 大于0.5 IU,结果为阳性;对狂犬病病毒中和抗体 (VNA) 小于0.5 IU/mL的血清,结果为阴性。制备的狂犬病病毒抗体胶体金检测试纸检测犬血清抗体,具有特异、便捷、快速的特点,能够检测出狂犬病病毒中和抗体大于0.5 IU的血清,适用于临床犬血清抗体水平监测,具有良好的应用前景。     

甲胎蛋白和癌胚抗原的胶体金免疫层析同步检测

目的:探讨建立一种同时快速检测甲胎蛋白(AFP)和癌胚抗原(CEA)两种肿瘤标志物的方法.方法:用胶体金分别标记鼠抗AFP单克隆抗体和鼠抗CEA单克隆抗体,喷于胶金垫上.与之配对的抗体喷在硝酸纤维素膜(NC膜)上分别做检测线T2和T1,兔抗鼠二抗喷在NC膜上做质控线,利用免疫层析技术进行检测.结果:采用Frens方法制备的胶体金粒径在20nm,紫外可见吸收峰在521 nm处,金标抗体的最适pH在9.0,最适抗体量为200μl,胶体金标记2.4μg抗体.测试结果表明同时检测AFP和CEA的试纸条灵敏度分别为20 ng.mL-1和5 ng.mL-1,检测时间仅需10min,与PSA、CA125、CA15-3、Ferritin、Human Albumin等没有交叉反应,稳定性好.结论:应用胶体金免疫层析技术,实现了AFP和CEA的同步检测.     

一种基于量子点检测抗CCP抗体的免疫荧光层析法

抗环瓜氨酸多肽 (cyclic citrullinated peptide, CCP) 抗体是类风湿关节炎 (rheumatoid arthritis, RA) 早期诊断的重要生物标志物. 为了实现对RA的早期诊断,本研究建立了一种基于CdTe量子点标记技术检测抗CCP抗体的免疫荧光层析法. 将CCP多肽与小牛血清白蛋白 (bovine serum albumin, BSA) 连接,再将CCP-BSA和 羊抗鼠IgG分别在硝酸纤维素膜 (nitrocellulose membrane, NC膜) 上划线,作为检测线 (test line, T线) 和质控线 (control line, C线). 制备量子点并在量子点上标记鼠抗人IgG,喷在玻璃纤维上并烘干,最后组装大卡、切割并封装制成检测试纸条. 应用该试纸条检测了RA患者及健康人血清临床样本200份,以酶联免疫吸附 测定法 (enzyme-linked immunosorbent assay, ELISA) 为对照,计算免疫荧光层析法的检测灵敏度和特异性. 结果显示,建立的量子点免疫荧光层析试纸条检测抗 CCP抗体的灵敏度为97.5 %,特异性为95.8%. 该方法操作简单、快速,可实现床旁检测 (point-of-care testing, POCT),能应用于RA的早期诊断.     

布鲁氏菌BP26抗原制备及基于BP26的金标检测试纸的初步研究

目的 制备布鲁氏菌血清学优势抗原BP26的原核表达,对其进行鉴定,且进行基于BP26重组蛋白的布鲁氏菌抗体金标检测试纸的研究。方法 检索人用疫苗株布鲁氏菌bp26基因序列进行基因合成,克隆至pET30a(+)载体,构建重组质粒pET30a(+)-bp26,转化大肠埃希菌BL21(DE3),IPTG诱导表达,亲和层析纯化BP26重组蛋白,纯化后重组蛋白进行蛋白免疫印迹鉴定。胶体金标记BP26,以BP26重组蛋白和BP26兔多抗血清分别包被检测线和质控线,制成布鲁氏菌抗体金标检测试纸。结果 BP26重组蛋白在大肠埃希菌中成功表达,相对分子质量约为26 000,与预期相符。亲和层析纯化后得到纯度较高的BP26重组蛋白。Western blot实验证明,该蛋白可与布鲁氏菌抗体阳性兔血清产生特异性结合。用布鲁氏菌抗体金标检测试纸检测兔阳性血清,检测线和质控线均显色明显。结论 成功获得BP26重组蛋白,Western blot实验证明该蛋白具有良好的免疫反应性。研制的检测试纸成功检测出布鲁氏菌抗体阳性兔血清。该蛋白为布鲁氏菌病诊断试剂盒研制及疫苗研发奠定了基础。     

三聚氰胺胶体金免疫层析试纸条的研制

通过胶体金免疫层析技术建立一种特异、便捷、快速的三聚氰胺抗原检测方法,对奶制品及饲料中的三聚氰胺残留水平监测提供参考。用柠檬酸三钠还原法制备的胶体金,标记纯化的三聚氰胺单克隆抗体,喷于试纸的金标垫。将MEL-OVA(三聚氰胺和卵清白蛋白的偶练物)和纯化的羊抗鼠IgG分别喷于试纸的T(检测线)处和C(质控线)处,通过挑选试纸条材料和调试工艺参数,并最终组装成试纸条。结果显示,制备的试纸监测体系方法检出限为50 g/L。试纸条对牛奶、奶粉和饲料中的三聚氰胺残留的检出限分别为100 g/L、100 ng/g和200 ng/g。将该法与LC-MS/MS法对比检测牛奶、奶粉和饲料样品,在试纸条检测范围内,与LC-MS/MS法检测结果一致性好,从而验证了该方法的有效性。制备的三聚氰胺胶体金检测试纸在常温干燥环境下至少可保质6个月,能够检测出三聚氰胺含量大于50 g/L的样品,适用于现场实际样品三聚氰胺残留水平监测,具有良好的应用前景。     

六种检测猪瘟病毒方法的比较

【目的】本研究旨在比较6种检测猪瘟病毒方法的优缺点。【方法】应用病毒分离、胶体金免疫层析试纸条、抗原捕捉ELISA、反转录-聚合酶链式反应(RT-PCR)、TaqMan荧光定量RT-PCR(RT-qPCR)和反转录-环介导等温扩增方法(RT-LAMP)等6种方法,分别对50份疑似猪瘟病料中的猪瘟病毒(Classical swine fevervirus,CSFV)进行检测。【结果】结果表明:RT-qPCR和RT-LAMP方法检出阳性样品数为13份,RT-PCR为11份,病毒分离为10份,抗原捕捉ELISA为9份,胶体金试纸条为8份;6种方法均检测为阳性8份,均为阴性37份。【结论】结果提示,在对猪瘟病毒进行检测时,RT-qPCR、RT-LAMP和RT-PCR由于其灵敏性高,可作为首选检测方法,但操作时需要避免假阳性的出现;病毒分离方法虽然操作繁琐,但结果准确,是确诊猪瘟必不可少的检测方法;抗原捕捉ELISA和胶体金试纸条检测时间较短,由于其敏感性较低所限,主要用于对畜群进行检测,不适合个体检测。     

基于N蛋白单克隆抗体的小反刍兽疫病毒抗体检测胶体金试纸条的研制

本研究旨在建立一种简便、快捷、可直观检测小反刍兽疫病毒(peste des petits ruminants virus,PPRV)抗体的检测方法。将pET-32a-N重组质粒转化至大肠杆菌(Escherichia coli) Rosetta(DE3)感受态细胞中进行诱导表达,以纯化的PPRVN蛋白免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,间接酶联免疫吸附试验(enzyme-linked immunosorbent assays, ELISA)筛选及亚克隆,获得了抗PPRV N蛋白的单克隆抗体。将PPRV N蛋白分别作为金标抗原及检测线(T线)包被抗原、单克隆抗体作为质控线(C线)包被抗体,组装成检测PPRVN蛋白抗体的胶体金免疫层析试纸条。结果显示：成功获得1株能稳定分泌抗N蛋白抗体的杂交瘤细胞株,命名为1F1;间接ELISA检测1F1腹水效价为1:128000;亚类鉴定结果为IgG1,轻链为kappa链。Westernblotting结果显示,1F1能与PPRV N蛋白特异性结合;间接免疫荧光(indirect immunofluorescent ass...     

心肌型脂肪酸结合蛋白胶体金检测试纸条的制备和初步应用

目的应用胶体金免疫层析法制备检测全血或血清样本中心肌型脂肪酸结合蛋白（H-FABP）的检测试纸条,用于急性心肌梗塞（AMI）的早期辅助诊断。方法采用柠檬酸三钠还原法制备胶体金,标记鼠抗心肌型脂肪酸结合蛋白单克隆抗体,喷于玻璃纤维膜上制成胶体金结合物垫,将另一株鼠抗心肌型脂肪酸结合蛋白单克隆抗体和抗鼠二抗分别包被检测线和质控线,组装成试纸条进行灵敏性、特异性、精密性、稳定性及临床样品检测。结果该试纸条的检测灵敏度为10ng／mL,15min内可判定结果;与肌钙蛋白I、C反应蛋白、肌酸激酶、人心肌肌红蛋白无交叉反应。检测240份临床标本,与临床诊断结果进行配对分析,阳性符合率95．83％、阴性符合率100％、总符合率97．92％。结论制备的H-FABP检测试纸条有良好的灵敏性、特异性,可用于早期AMI的辅助诊断。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.