一种快速检测弗氏柠檬酸杆菌免疫层析试纸条的研制
PDF下载 (521)孙智同,章礼平,李登峰.一种快速检测弗氏柠檬酸杆菌免疫层析试纸条的研制[J].宁波大学学报(理工版),2019,32(3):15-20.DOI:
SUN Zhi-tong,ZHANG Li-ping,LI Deng-feng*.Development of a colloidal gold immunochromatographic test strip for detection of Citrobacter freundii[J].Journal of Ningbo University(Natural Science & Engineering Edition),2019,32(3):15-20.DOI:
| Title: | Development of a colloidal gold immunochromatographic test strip for detection of Citrobacter freundii |
| 作者: | 孙智同, 章礼平, 李登峰 |
| Author(s): | SUN Zhi-tong, ZHANG Li-ping, LI Deng-feng* |
| 关键词: | 弗氏柠檬酸杆菌 ; 卵黄抗体; 胶体金; 免疫层析试纸; 检测 |
| Keywords: | Citrobacter freundii; IgY; colloidal gold; immunochromatographic strip; detection |
| 分类号: | Q93-331 |
| 文献标识码: | A |
| 摘要: | 为了制备弗氏柠檬酸杆菌快速检测试纸条, 联合卵黄抗体技术和胶体金免疫层析技术, 采用弗氏柠檬酸杆菌抗原免疫母鸡, 制备抗弗氏柠檬酸杆菌卵黄抗体(IgY); 采用柠檬酸三钠还原法制备胶体金颗粒, 用以标记IgY; 在结合垫上包被用胶体金标记的IgY, 硝酸纤维膜上包被弗氏柠檬酸杆菌抗原和羊抗鸡IgY作为检测线和质控线; 将样品垫、金标结合垫、硝酸纤维膜和吸水垫组装成试纸条, 测试其灵敏度、特异性、稳定性, 并与ELISA结果进行比较. 结果表明: 该试纸条检测线为105 cfu∙mL-1; 试纸条与恶臭假单胞菌、铜绿假单胞菌、迟钝爱德华菌、哈维氏弧菌和溶藻弧菌均无交叉反应; 该试纸条在4℃保存4个月后依然十分稳定; 具有易操作, 特异性、灵敏度高, 不需要专业的操作人员和特殊的仪器设备, 5~10min即可获得结果等优势, 适合弗氏柠檬酸杆菌的现场检测. |
| Abstract: | The current study was aimed to establish a rapid test strip for Citrobacter freundii by using egg yolk antibody and colloidal gold immunochromatography assay. Hens were immuned with C. freundii antigen. Antibody against C. freundii was purified from the yolks. The colloidal gold particles combining with yolk antibodies (IgY) were prepared by citric acid three sodium reduction method. The labeled colloidal gold-IgY complex was wrapped on the binding pad, and the nitric acid fiber membrane was coated with C. freundii antigen and Sheep anti-chicken IgY as the test line and the control line respectively. The sample pad, gold conjugate pad, nitrocellulose membrane and absorbent pad were assembled into test strips and sealed with desiccant. The sensitivity, specificity and stability of the test strip were tested, and test results were compared with those from ELISA. The detection results can be obtained within 5-10min. The sensitivity of the test strip was 105 cfu?mL-1, and there was no cross reaction with Pseudomonas putida, P. aeruginosa, Edwardsiella tarda, Vibrio harveyi and V. alginolyticus. The strip was stable and efficient after being stored at 4℃ for 4 months. As detection with the strip is easy to operate and does not require professional operator and special instrument, it is very suitable for the on-site detection of C. freundii |
| 参考文献 /References: | [1] Guerrant R L, Dickens M D, Wenzel R P, et al. Toxigenic bacterial diarrhea: Nursery outbreak involving multiple bacterial strains[J]. Journal of Pediatrics, 1976, 89:885- 891. [2] Badger J L, Stins M F, Kim K S. Citrobacter freundii invades and replicates in human brain microvascular endothelial cells[J]. Infection and Immunity, 1999, 67(8): 4208-4215. [3] Gupta N, Yadav A, Choudhary U, et al. Citrobacter bacteremia in a tertiary care hospital[J]. Scandinavian Journal of Infectious Diseases, 2003, 35:765-768. [4] Bai L, Xia S L, Lan R T, et al. Isolation and characterization of cytotoxic, aggregative Citrobacter freundii[EB/OL]. [2018-05-10]. https://doi.org/10.1371/ journal.pone.0033054. [5] Dervisoglu E, Yumuk Z I. Citrobacter freundii peritonitis and tunnel infection in a patient on continuous ambulatory peritoneal dialysis[J]. Journal of Medical Microbiology, 2008, 57(1):125-127. [6] Tschape H, Prager R, Streckel W, et al. Verotoxinogenic Citrobacter freundii associated with severe gastroenteritis and cases of haemolytic uraemic syndrome in a nursery school: Green butter as the infection source[J]. Epidemiology Infection, 1995, 114:441-450. [7] Warner R D, Carr R W, McCleskey F K, et al. A large nontypical outbreak of Norwalk virus. Gastroenteritis associated with exposing celery to nonpotable water and with Citrobacter freundii[J]. Archives of Internal Medicine, 1991, 151:2419-2424. [8] Parida S N, Verma I C, Deb M, et al. An outbreak of diarrhea due to Citrobacter freundii in a neonatal special care nursery[J]. Indian Journal of Pediatrics, 1980, 47: 81-84. [9] Nada T, Baba H, Kawamura K, et al. A small outbreak of third generation cephem-resistant Citrobacter freundii infection on a surgical ward[J]. Japanese Journal of Infectious Diseases, 2004, 57(4):181-182. [10] Galarneau J R, Fortin M, Lapointe J M, et al. Citrobacter freundii septicemia in two dogs[J]. Journal of Veterinary Diagnostic Investigation, 2003, 15(3):297-299. [11] Joaquin A, Khan S, Russel N, et al. Neonatal meningitis and bilateral cerebellar abscesses due to Citrobacter freundii[J]. Pediatric Neurosurgery, 1991, 17:23-24. [12] 郑善坚. 弗氏柠檬酸杆菌引起鱼、蛙溃烂病的防治案例[J]. 科学养鱼, 2017(2):68-69. [13] Bae J Y, Kang C K, Choi S J, et al. Sudden deaths of neonates receiving intravenous infusion of lipid emulsion contaminated with Citrobacter freundii[EB/OL]. [2018- 05-10]. https://doi.org/10.3346/jkms.2018.33.e97. [14] Liu H, Zhao Z, Xue Y, et al. Fatal cases of Citrobacter freundii septicemia and encephalitis in sheep[J]. Journal of Veterinary Diagnostic Investigation, 2018, 30(2): 245-248. [15] 闫革彬, 隋吉林, 芦丹, 等. 一起弗氏柠檬酸杆菌所致食源性疾病调查[J]. 疾病检测, 2013, 28(3):247-248. [16] 王金洛, 徐福州. 卵黄抗体研究进展[J]. 农业新技术, 2003(6):36-38. [17] 周永强, 李登峰, 刘联国, 等. 抗鳗利斯顿氏菌鸡卵黄抗体制备及其活性研究[J]. 宁波大学学报(理工版), 2012, 25(4):1-5. [18] Frens G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions[J]. Nature Physical Science, 1973, 241:20-22. [19] Zhang L P, Li D F, Liu L G, et al. A rapid immunochromatographic test strip for detecting swimming crab (Portunus trituberculatus) reovirus[J]. Disease of Aquatic Organism, 2015, 117:21-29. [20] Zhang L P, Li D F, Liu L G, et al. Development of colloidal gold immunochromatographic strip for rapid detection of soft-shelled turtle systemic septicemia spherical virus[J]. Journal of Virological Methods, 2015, 221:39-45. [21] Schade R, Calzado E G, Sarmiento R, et al. Chicken egg yolk antibodies (IgY-technology): A review of progress in production and use in research and human and veterinary medicine[J]. Atla-alternatives to Laboratory Animals, 2005, 33:129-154. [22] Davalos-Pantoja L, Ortega-Vinuesa J L, Bastos-Gonzalez D, et al. A comparative study between the adsorption of IgY and IgG on latex particles[J]. Journal of Biomaterials Science-Polymer Edition, 2000, 11(6):657-673. |
| 备注/Memo: | 收稿日期: 2018?07?10. 宁波大学学报（理工版）网址: http://journallg.nbu.edu.cn/ 基金项目: 国家海洋局开放基金（HY201602）; 宁波大学王宽诚幸福基金. 第一作者: 孙智同（1993－）, 男, 浙江宁波人, 在读硕士研究生, 主要研究方向: 水产动物病害防治. E-mail: 1611075773@nbu.edu.cn *通信作者: 李登峰（http://journallg.nbu.edu.cn/ |
