布鲁氏菌抗体胶体金免疫层析法快速检测技术的建立

目的建立一种快速检测布鲁氏菌抗体的新方法。方法利用胶体金免疫层析技术,以大肠埃希菌表达、纯化的OMP31与BP26重组蛋白作为检测抗原,以金黄葡萄球菌A蛋白(SPA)作为胶体金标记物,制备胶体金免疫层析试纸条,用于检测布鲁氏菌抗体,并分析方法的敏感性和特异性。结果以粒径为40nm胶体金制备的试纸条检测布鲁氏菌抗体的敏感性最高,胶体金最佳标记pH为6.2,SPA蛋白最适标记量为6μg/ml。交叉试验证明试纸条不与其他非相关疾病感染血清反应,特异性高。试纸条检测结果与琥红平板试验方法的符合率为92%。结论制备的布鲁氏菌抗体胶体金免疫层析试纸条具有敏感、特异、简便、快速的特点,可用于鉴别布鲁氏菌自然感染和人工免疫,并可区分牛、羊种布鲁氏菌感染,可在基层推广使用。     

人卡他莫拉单克隆抗体胶体金试纸的制备及初步应用

目的制备抗UspA1蛋白单克隆抗体,研制能快速、灵敏、准确检测卡他莫拉菌的胶体金免疫层析试纸,以期用于人卡他莫拉菌的临床检测。方法将重组UspA1-His蛋白作为抗原,免疫小鼠,并制备单克隆抗体;通过Western blot技术以及免疫荧光技术鉴定抗体的特异性;以双抗体夹心的原理研制胶体金免疫层析检测试纸条,并对其特异性、敏感性及稳定性进行评价分析。结果筛选出两株分泌特异性抗体的杂交瘤细胞株,经腹水制备纯化得到单克隆抗体,并验证了两种抗体均能特异性识别天然UspA1蛋白;利用双抗夹心原理制备的胶体金试纸能在10 min内快速检测卡他莫拉菌,检测灵敏度高(1×10~5 CFU/ml)、特异性强,与其他常见9种的呼吸道病原菌诸如肺炎链球菌、流感嗜血杆菌、肺炎支原体、嗜肺军团菌等无交叉反应;试纸条在37℃保存有良好的重复性和稳定性;200份临床样品检测结果与平板培养法的阳性符合率为93.3%。结论本研究制备了特异性强的抗UspA1蛋白单克隆抗体;以此研制的胶体金免疫层析试纸可快速、简便、灵敏的检测卡他莫拉菌,适用于呼吸道感染的临床快速检测。     

快速诊断滴虫性阴道炎免疫层析试纸条的初步研制

目的 用胶体金标记抗阴道毛滴虫重组AP33单克隆抗体制备金标AP33 mAb,研制开发出一种能快速、简便、有效检测阴道毛滴虫感染的免疫层析（GICA）试纸条。方法 采用柠檬酸钠还原法制备胶体金颗粒,标记抗阴道毛滴虫重组AP33单克隆抗体,选择最佳反应模式组装试纸条。用该试纸条检测滴虫性阴道炎患者及非滴虫性阴道炎患者白带中的靶抗原,鉴定方法的敏感性和特异性。结果 用制备的免疫层析试纸条检测重组AP33抗原的最低量为20 ng/ml,其中以单克隆抗体4A2为包被抗体、单克隆抗体4F8为金标抗体的试纸条检测效果最佳。以湿片法为标准,用该试纸条检测60例滴虫性阴道炎患者白带,敏感性为90.0%（54/60）;检测60例非滴虫性阴道炎患者白带,特异性为95.0%（57/60）。两法的总符合率为92.5%。结论 GICA检测阴道毛滴虫特异性强、灵敏度高、简便快速,无需特殊仪器设备,有广泛应用价值。     

猪带绦虫六钩蚴TSOL18重组蛋白快速诊断试纸条研制

目的以猪带绦虫六钩蚴重组蛋白(TSOL18)为抗原,建立一种简便快速的猪囊虫抗体检测方法,评价其血清学诊断的价值。方法用胶体金颗粒标记纯化的TSOL18重组蛋白,将兔抗TSOL18-GST-IgG和TSOL18重组抗原分别喷涂于硝酸纤维素膜的质控线和检测线上,与PVC垫板等部件按顺序装配成猪囊虫抗体快速检测试纸条;用该试纸条检测猪血清样品测定敏感性和特异性。结果该试纸条与全囊虫抗原ELISA的相对敏感性和特异性分别为75.0%(12/16)和95.2%(40/42),与剖检计数法的相对敏感性达80.0%(12/15)。试纸条在4℃存放12个月,检测结果稳定,重复性好。结论基于TSOL18建立的胶体金免疫层析试纸条操作简单、快速敏感、稳定性好,可用于猪囊虫病感染的诊断和筛查。     

胶体金免疫层析检测犬、猫抗狂犬病病毒IgG抗体的研究

目的建立检测犬、猫抗狂犬病病毒(RV)IgG抗体的胶体金免疫层析方法。方法采用柠檬酸三钠还原法制备胶体金用以标记SPA,同时将重组RVN蛋白、抗SPA抗体分别包被至硝酸纤维素膜的检测线与质控线上,制备一种检测犬、猫抗RVIgG抗体的胶体金检测卡,进行了特异性和灵敏度试验,并与ELISA同时检测临床样品、统计结果。结果 GICA试纸条检测灵敏度为0.5IU/mL,与犬瘟热病毒、犬细小病毒等阳性血清无交叉反应,并与ELISA相比,两者的符合率为94.6%。结论成功建立了检测犬、猫抗RVIgG抗体的通用型胶体金免疫层析方法 ,该方法灵敏度高,特异性强,检测速度快,操作简便,可广泛应用于基层。     

检测鼠疫抗体胶体金免疫层析试纸条的研制

目的建立快速、简易的检测鼠疫F1抗体的胶体金免疫层析法(G ICA)。方法(1)采用胶体金颗粒标记纯化F1抗原,并将标记物喷于玻璃纤维;同时将纯化F1抗原喷线固定于硝酸纤维素膜上,用于F1抗体的捕捉;按常规组装成检测鼠疫抗体免疫层析试纸条。(2)采用该试纸条与血凝法对同一份兔抗F1抗体进行检测,以评价该试纸条的敏感性。(3)采用该试纸条对44株非鼠疫菌的免疫鼠血清进行检测,以评价该试纸条的特异性。(4)采用该试纸条、血凝法及ELISA对607份血清标本进行检测,以评价该试纸条对现场材料的检测效果。结果(1)该试纸条可在15 m in之内完成检测;(2)在敏感性上,该试纸条对同一份免疫兔血清的检测较血凝法高一个滴度;(3)对被试的44株所选菌株的免疫鼠血清的检测均为阴性;(4)在对607份血清标本的检测中,免疫层析试纸条、血凝及ELISA三种方法的符合率中度,而免疫层析试纸条的敏感性分别比血凝与ELISA高111%和90%。结论以纯化的鼠疫F1抗原为基础建立的G ICA检测鼠疫F1抗体的方法特异性强、灵敏度高、简便快速,无需特殊仪器设备,有较大的推广应用价值。     

霍乱弧菌O139胶体金免疫层析快速检测法的建立

摘 要：目的 建立一种快速、简易的检测霍乱弧菌O139群的胶体金免疫层析法（GICA）。方法 利用胶体金免疫层析技术, 采用双抗体夹心法检测霍乱弧菌O139群, 对该法进行敏感性、特异性和稳定性分析。与细菌培养法对比检测184 份临床标本。结果 该法能在10 分钟内完成检测;该试纸条仅与霍乱弧菌O139群阳性样品发生特异性反应;检测霍乱弧菌O139群的最低检出浓度为1×105cfu/mL;与细菌培养法对比检测184 份临床标本, 特异性和灵敏度均达 100%。结论 新建立的霍乱弧菌O139群胶体金免疫层析试验简便、快速, 特异性和灵敏度较好, 适用于现场样品的快速筛查。     

胶体金标记免疫层析法检测O∶9型小肠结肠炎耶尔森菌

目的建立一种简便快速的胶体金标记免疫层析(GICA)条用于检测标O∶9血清型小肠结肠炎耶尔森氏菌。方法采用柠檬酸三钠还原法制备胶体金颗粒,标记抗小肠结肠炎耶尔森菌单克隆抗体,以硝酸纤维膜作为抗小肠结肠炎耶尔森氏菌单克隆抗体的包被载体,制成GICA检测条。被检小肠结肠炎耶尔森菌与检测卡上金标记抗体(Au-Ab)结合后,利用硝酸纤维膜的层析作用,与膜上的固相抗体结合形成可见的红色条带。结果GICA小肠结肠炎耶尔森菌检测条灵敏度可达104~105CFU/ml,与其他所选择肠杆菌科细菌均未发现交叉反应,仅与布鲁菌出现交叉。检测了50株O∶9血清型小肠结肠炎耶尔森氏菌,符合率为100%。结论GICA小肠结肠炎耶尔森菌检测条对小肠结肠炎耶尔森菌特异性强;简便快速,不需任何仪器设备;结果易于判断,可望用于标本快速筛查。     

基于微波法快速合成胶体金建立猴痘病毒抗原免疫层析试纸条半定量方法

目的 建立猴痘病毒(Monkeypox virus, MPXV)A35R蛋白现场快速半定量检测胶体金免疫层析方法。方法 以微波法6 min快速合成金纳米颗粒(Gold nanoparticles, AuNPs)作为标记物，基于双抗体夹心法体系建立检测MPXV A35R蛋白的免疫层析试纸方法。利用真核系统表达的MPXV包膜成分A35R蛋白配制系列浓度标准液，初步评价胶体金免疫层析试纸条的敏感性、特异性及稳定性等检测性能。结果 20 min内完成检测，建立MPXV抗原的胶体金免疫层析试纸的半定量检测的灵敏度达到62.5 pg/mL,肉眼最低检测限是125 pg/mL。与登革病毒(Dengue virus, DENV)、诺如病毒(Norovirus, NV)均无交叉反应，稳定性良好。结论 本研究建立的胶体金半定量免疫层析试纸条可以实现对MPXV的快速灵敏检测，在MPXV的即时检测(Point of care testing, POCT)领域具有一定潜力。     

肠出血性大肠埃希菌O157∶H7环介导恒温扩增快速检测方法的建立与应用

目的肠出血性大肠埃希菌(Enterohemorrhagic Escherichia coli,EHEC)O157∶H7是一种重要的人畜共患病致病菌,主要通过被污染的食物传播,引起出血性结肠炎,建立其快速检测方法具有重要意义。方法基于EHEC O157∶H7保守的rfbE基因序列,设计4条引物,利用环介导等温扩增技术(loop-mediatedisothermal amplification,LAMP),成功建立了EHEC O157∶H7LAMP快速检测方法 ,60min内即可完成致病菌检测。结果利用该LAMP方法对30种共38株细菌进行检测,所试EHEC O157∶H7均为阳性结果 ,说明该方法具有高度特异性。本方法对纯培养的EHEC O157∶H7检测限为12CFU/mL,污染食品中EHEC O157∶H7的检测限为18CFU/g。实践应用表明,对1121份进出口肉类、奶类制品及人工污染样品等进行检测,检出57份LAMP阳性,与采用AOAC标准检测结果的符合率为100%。结论该LAMP方法操作简便、特异性强、灵敏度高,具有良好的实用性。     

抗出血性大肠杆菌Ο157∶H7单克隆抗体的制备及鉴定

目的制备抗出血性大肠杆菌Ο157∶H7(E.coliΟ157∶H7)特异性单克隆抗体(MAbs)。方法福尔马林灭活的E.coliΟ157∶H7免疫BALB/c小鼠,利用细胞融合技术建立分泌抗E.coliΟ157∶H7MAbs的杂交瘤细胞株,对配对较好的6株MAbs用ELISA法测定其免疫球蛋白类及亚类,用ELISA法、凝集法和Westernblot鉴定MAbs的特异性。结果6株MAbs免疫球蛋白均为小鼠IgM。这些MAbs均能与27个E.coliΟ157∶H7菌株发生凝集反应,与部分弗劳地杆菌发生凝集反应,与11株鼠伤寒沙门氏菌、7株伤寒杆菌、2株痢疾杆菌、致病性大肠杆菌、产毒性大肠杆菌、侵袭性大肠杆菌、出血性大肠杆菌Ο26∶H11和Ο111、肠集聚性大肠杆菌、42株非定血清型大肠杆菌、霍乱弧菌Ο1群和Ο139群不发生凝集反应;ELISA结果显示6株MAbs与粪链球菌、变形杆菌、粘质沙雷氏菌、肺炎克雷伯杆菌均无交叉反应;ELISA和Westernblot结果显示,3株MAbs针对E.coliΟ157∶H77酚相脂多糖。结论6株MAbs具有较高的特异性,有可能用于制备检测E.coliΟ157∶H7的病原检测试剂。     

Characterization of enterohemorrhagic Escherichia coli O26 and development of its isolation media]

We studied 101 strains of Enterohemorrhagic Escherichia coli (EHEC) O26 isolated from diarrhea patients in six prefectural institutes of public health in Japan during June 1996 and December 1997 and tried to establish an isolation medium for EHEC O26. None of the 101 EHEC O26 strains fermented rhamnose; Whereas all of the other EHEC including O157 and non-EHEC (166 strains) fermented rhamnose except 1 strain of non-EHEC. All of the randomly selected EHEC O26 (14 strains of O26:H11.2 strains of O26:H-) showed a very high resistance to potassium tellurite (Minimal Inhibitory Concentration (MIC) > or = 50 micrograms/ml), whereas all of the randomly selected non-EHEC (26 strains) but 1 showed a high sensitivity (MIC < or = 6.25 micrograms/ml) to this compound. On the basis of these results, we developed a Rhamnose MacConkey (RMAC) medium in which lactose in the MacConkey medium was replaced by rhamnose, and Cefixime-Potassium Tellurite-RMAC (CT-RMAC) medium in which Cefixime (0.05mg/l) and Potassium Tellurite (25mg/l) was added to RMAC for the isolation of EHEC O26 strains. We then evaluated the specifcity of these selective media by growing a selected number of O26 (24 strains) and 9 selected strains of bacteria. All of the EHEC O26 strains generated rhamnose non-fermented colonies (white color) on both media. In contrast to the EHEC O26, the vast majority of E. coli strains (166/167 = 99.4%) other than EHEC O26 were theoretically assumed to generate red colonies on the RMAC medium because of their rhamnose fermenting character and most of them were assumed not to grow on CT-RMAC medium because of their sensitivity to potassium tellurite. These findings and results indicate that EHEC O26 can be easily distinguished from other strains of E. coli including O157. Although EHEC O26 strains showed somewhat poor growth on CT-RMAC medium compared with that on RMAC medium, these O26 showed almost the same degree of growth on CT-RMAC as they showed on DHL media. The results of the present study demonstrated that the use of RMAC and CT-MRAC media for the isolation of EHEC O26 is very reliable and efficient with RMAC having good sensitivity and CT-RMAC having a better specificity for the isolation of this strain of EHEC.     

免疫捕获PCR法快速检测大肠埃希氏O157∶H7的研究

目的建立两种免疫捕获PCR法快速检测E.coliO157∶H7,并探讨其灵敏度和特异性。方法运用免疫磁珠集菌(IMS)和抗体包被扩增管(mACT)两种方法富集待测样品中E.coliO157∶H7,再应用巢式PCR法检测编码E.coliO157∶H7O抗原的rfbE基因,对13株E.coliO157菌和10株非E.coliO157菌进行检测。结果应用两种icPCR检测,所有E.coliO157∶H7和E.coliO157NM菌株均为阳性结果,而其它包括与O157抗血清能交叉凝集的菌株检测结果均为阴性;对于含有48×102cfu/ml以上浓度的E.coliO157∶H7菌悬液,两种方法,所有试验管都能检测到特异性扩增;并证实了这两种方法可用于检测鲜牛奶中E.coliO157∶H7,样品中E.coliO157∶H7的含量只要不少于101cfu/ml,经过6小时增菌均能检测出来。结论IMS-icPCR和mACT-icPCR用于检测E.coliO157∶H7,是快速、灵敏且高特异的方法。     

Escherichia coli 'O' group serology of a haemolytic uraemic syndrome (HUS) epidemic

This is the first comprehensive serological analysis of a haemolytic uraemic syndrome (HUS) outbreak. A wide range of 'O' group Escherichia coli antibody responses in patients and controls was examined. The study provides a unique insight into the epidemiology of such epidemics, points a way to the most appropriate investigation of these and indicates possible answers to a number of issues related to severity of disease. In order to be able to test for a wide variety of E. coli 'O' antigens, a microagglutination assay was used to examine E. coli 'O' group serological responses of 22 children admitted to hospital with HUS and 14 contemporaneous age-matched controls. A total of 51 'O' serogroup strains were used. These included 'O' groups reported to be associated with cases of HUS, with 6 isolates from patients associated with the Adelaide outbreak (O26, O111, O123 and O157), environmental Verocytotoxigenic/Shiga-toxin producing Escherichia coli (VTEC/STEC) strains and common human commensal strains. Sixteen clinically confirmed HUS cases (72.7%) of 22 seroconverted to 1 or more serogroups of which 11 (50%) seroconverted to O111 (the serogroup isolated from 16 patients). In addition, 11 (50%) and 10 (45.5%) developed antibody to O137 and O145, respectively, although no stool isolates of these serogroups were made. Seventeen (77.3%) of 22 HUS patients had antibody to serogroup O157, with 11 (50%) seroconversions, however, O157:H- was isolated from only 2 of these. Overall, titres ranged from 100 to 6400, some of the highest in 3 patients were against O157, whose faeces yielded only Enterohaemorrhagic E. coli (EHEC) O111, and only 1 developed O111 antibody. Mixed infection was demonstrated serologically by microagglutination (confirmed by Western blot) and was consistent with the findings of multiple serogroups of VTEC found in the mettwurst incriminated as the source, and suggests further strains (not found in the source or in patients' faeces) were probably also involved. In HUS associated with EHEC infection, multiple strain infection may be the rule rather than the exception. A relationship with clinical severity deserves further investigation. Non-O157 EHEC (in addition to O157) should be sought in all future outbreaks of EHEC disease.     

Serum anti-lPS antibody production in an outbreak of enterohemorrhagic Escherichia coli O157 infection among schoolchildren

Although there have been many reports of the usefulness of serodiagnosis of enterohemorrhagic Escherichia coli (EHEC) O157, the serotype of the bacteria detected and the increase in anti-LPS antibody have not always been consistent. In this study we investigated the diagnostic significance of measurements of anti-LPS antibody by ELISA in an outbreak of O157 infection among schoolchildren in whom the bacteriological test findings were clarified and the age groups were uniform. The anti-LPS antibody titer was measured in 31 patients (77 serum samples) in an outbreak of EHEC O157 : H7 infection (220 children infected) that occurred in a primary school in Morioka in 1996. The anti-O157 LPS antibody positivity rates of IgM, IgG, and IgA were 98.7%, 85.7%, and 98.7%, respectively. Between the time the meal that caused the outbreak and 19 days later, anti-O157 LPS IgM antibody and IgA antibody were detected in all patients. The specificity was investigated using control serum, and the specificity of IgM, IgG, and IgA was 93.5%, 93.5%, and 97.2%, respectively. Some samples contained antibodies against O111 and O26 LPS, but the titers were lower than the anti-O157 antibody titer. The anti-O111 antibody titer and anti-O26 antibody titer were highly correlated, suggesting that they were crossreactive antibodies for O157 LPS. No significant correlation was found between differences in clinical manifestations and the anti-O157 LPS antibody titer in this O157 outbreak in schoolchildren. It was clarified that an increase in anti-LPS antibody was found to support the diagnosis of mild cases of 0157 infection infection as well as severe cases.     

A DNA probe to identify enterohemorrhagic Escherichia coli of O157:H7 and other serotypes that cause hemorrhagic colitis and hemolytic uremic syndrome

Enterohemorrhagic Escherichia coli (EHEC) cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), make potent cytotoxins (Verotoxins [VT] or Shiga-like toxins), and possess a plasmid (approximately 60 megadaltons) that encodes a new fimbrial antigen and promotes attachment to epithelial cells. We evaluated the use of a DNA probe, prepared from a 3.4-kilobase segment of the EHEC plasmid, to detect EHEC. The probe hybridized with 106 (99%) of 107 O157:H7 and 34 (77%) of 44 O26:H11, VT-positive strains from patients with colitis, HUS, and diarrheal disease and hybridized with 21 (81%) of 26 VT-positive E. coli of serotypes other than O157:H7 or O26:H11 from patients with hemorrhagic colitis and HUS. We examined 601 other strains, including 18 serotype O26 isolates of H types other than H11, 306 enteropathogenic E. coli, 60 enteroinvasive E. coli, 119 enterotoxigenic E. coli, and 20 isolates from the urinary tract and 77 isolates from the normal intestinal flora; only one (O127:H-) was positive (specificity, 99.8%). Serotype O26:H11, previously considered a classic enteropathogenic E. coli serotype, is now shown to be EHEC.     

Molecular characterization of enterohemorrhagic Escherichia coli O157:H7 isolates dispersed across Japan by pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis

We identified seven distinct subtypes of enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were derived from sporadic cases and outbreaks from multiple prefectures in Japan in 2005. A surveillance system utilizing pulsed-field gel electrophoresis (PFGE), PulseNet Japan, was used. Some strains showed indistinguishable PFGE patterns using another restriction enzyme (BlnI or SpeI) in each subtype of EHEC O157:H7 isolates that were routinely subtyped by the XbaI PFGE pattern. In order to examine the genotypic relatedness of these strains, we carried out a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA). By using the MLVA system, we found that three of seven subtypes of EHEC O157:H7 strains that were isolated from sporadic cases dispersed across multiple prefectures within a few months showed indistinguishable PFGE patterns and identical MLVA types. Strains belonging to the other four subtypes of EHEC O157:H7 in the PFGE analysis were further classified into different clusters of EHEC O157:H7. Therefore, compared to PFGE, MLVA showed greater discriminatory power with respect to analysis of the isolates in this study.     

Rapid and simple method for the detection of shigatoxins (STxs)

Enterohemorrhagic Escherichia coli (EHEC) is an emerged bacterial agent as the cause of bloody diarrhea and a leading cause of hemolytic uremic syndrome in children. In our country, serotype O 157:H7 is the predominant pathogen in the EHEC and the most frequently seen in human infections. The clinical disease is not associated with the STx types produced by the infecting strains. Non-O157 serotypes of EHEC also produce STxs, and infections with some non-O157 EHEC are occasionally associated with the illness caused by O157:H7. A rapid and simple enzyme immunoassay method (EIA) for the detection of STxs was established. This method is based on the use of anti-STx1 or anti-STx2 monoclonal antibody-labelled colloidal gold for detective factor and also used each anti-STx antibody for the capture antibody. The supernatant was used as the test sample after centrifugation of bacterial suspension treated with polymixin B (5,000 u/ml). These EIA-tests were specific for all supernatants of EHEC serotypes used, giving positive reactions (more than 1:16-32) by the RPLA method, and permitted the detection of ca. 203-812 pg of STx1 or STx2 in a 130 microliters applied to this test. This method will be an extremely useful tool for the detection of STxs from isolates or bacteria on selective agar-plates.     

肠出血性大肠杆菌O157∶H7 TccP基因克隆、表达及其抗原性鉴定

目的克隆表达肠出血性大肠杆菌(EHEC)O157∶H7 Tir细胞骨架偶联蛋白(TccP)基因,并研究其抗原性。方法采用PCR法自EHEC O157∶H7 Sakai菌株基因组扩增TccP基因,构建TccP原核表达载体并在大肠杆菌中诱导表达。表达产物经初步纯化后,免疫新西兰白兔,制备兔抗TccP多克隆抗体。抗体效价及特异性用ELISA法和Western blotting法进行测定和分析。结果从EHEC O157∶H7 Sakai菌株中克隆出了1014bp的TccP基因。构建的重组质粒pET28a-TccP在大肠杆菌中获得了高效表达;以TccP免疫家兔获得了高效价多克隆抗体,Western blotting分析此抗体能与TccP发生特异性抗原抗体反应。结论在大肠杆菌中成功克隆表达了TccP基因,所获TccP具有良好的抗原性,为进一步研究TccP在EHEC O157∶H7致病机制中的作用奠定基础。