Hou Wanwan, Wang Shaohui, Wang Xiaolan, Han Xiangan, Fan Hongjie, Cao Shoulin, Yue Jiaping, Wang Quan, Jiang Wei, Ding Chan, Yu Shengqing
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China; Key Lab of Animal Bacteriology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China.
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
PLoS One. 2015 Mar 30;10(3):e0122952. doi: 10.1371/journal.pone.0122952. eCollection 2015.
Riemerella anatipestifer is one of the most important bacterial pathogen of ducks and causes a contagious septicemia. R. anatipestifer infection causes serositis syndromes similar to other bacterial infections in ducks, including infection by Escherichia coli, Salmonella enterica and Pasteurella multocida. Clinically differentiating R. anatipestifer infections from other bacterial pathogen infections is usually difficult. In this study, MAb 1G2F10, a monoclonal antibody against R. anatipestifer GroEL, was used to develop a colloidal gold immunochromatographic strip. Colloidal gold particles were prepared by chemical synthesis to an average diameter of 20 ± 5.26 nm by transmission electron microscope imaging. MAb 1G2F10 was conjugated to colloidal gold particles and the formation of antibody-colloidal gold conjugates was monitored by UV/Vis spectroscopy. Immunochromatographic strips were assembled in regular sequence through different accessories sticked on PVC plate. Strips specifically detected R. anatipestifer within 10 min, but did not detect E. coli, S. enterica and P. multocida. The detection limit for R. anatipestifer was 1 × 10(6) colony forming units, which was 500 times higher than a conventional agglutination test. Accuracy was 100% match to multiplex PCR. Assay stability and reproducibility were excellent after storage at 4°C for 6 months. The immunochromatographic strips prepared in this study offer a specific, sensitive, and rapid detection method for R. anatipestifer, which is of great importance for the prevention and control of R. anatipestifer infections.
鸭疫里默氏菌是鸭最重要的细菌病原体之一,可引起传染性败血症。鸭疫里默氏菌感染会导致与鸭的其他细菌感染相似的浆膜炎综合征,包括大肠杆菌、肠炎沙门氏菌和多杀性巴氏杆菌感染。临床上通常很难将鸭疫里默氏菌感染与其他细菌病原体感染区分开来。在本研究中,使用抗鸭疫里默氏菌GroEL的单克隆抗体1G2F10开发了一种胶体金免疫层析试纸条。通过化学合成制备胶体金颗粒,通过透射电子显微镜成像,其平均直径为20±5.26nm。将单克隆抗体1G2F10与胶体金颗粒偶联,并通过紫外/可见光谱监测抗体-胶体金偶联物的形成。通过粘贴在PVC板上的不同配件按常规顺序组装免疫层析试纸条。试纸条在10分钟内可特异性检测鸭疫里默氏菌,但未检测到大肠杆菌、肠炎沙门氏菌和多杀性巴氏杆菌。鸭疫里默氏菌的检测限为1×10(6)菌落形成单位,比传统凝集试验高500倍。准确性与多重PCR的匹配率为100%。在4°C下储存6个月后,检测方法的稳定性和重现性良好。本研究制备的免疫层析试纸条为鸭疫里默氏菌提供了一种特异性强、灵敏度高、快速的检测方法,对鸭疫里默氏菌感染的预防和控制具有重要意义。