口蹄疫病毒通用型金标检测试纸条诊断方法的建立  

Development of a Rapid Gold Immunochromatographic Strip Test for the Diagnosis of Foot-and-Mouth Disease Virus

作　　者：任维维[1,2] 梁仲[1] 智晓莹[1,2] 祁光宇[1,2] 刘湘涛[1] 才学鹏[1] 蒋韬[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室/国家口蹄疫参考实验室,兰州730046 [2]中农威特生物科技股份有限公司,兰州730046

出　　处：《畜牧兽医学报》2012年第2期255-262,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：甘肃省科技厅资助;甘肃科技重大技术专项(1002NKDA037)

摘　　要：为了兽医基层工作人员能够快速诊断口蹄疫病原,与其他传染病做鉴别诊断,有必要建立一种快速、简便区分它们的胶体金免疫层析方法。本试验采用柠檬酸三钠还原法制备胶体金。将纯化的Asia 1/China/05流行毒株12S偶联抗原的兔抗体制备免疫胶体金,喷免疫胶体金于玻璃纤维制成金标垫。将纯化A/AF72流行株12S偶联抗原的兔抗体和适量O/China/99流行毒株12S偶联抗原的兔抗体混合固定于硝酸纤维素膜上作为检测带。将羊抗兔抗体固定在硝酸纤维素膜的不同区域作为质控带,并组装成口蹄疫病毒通用型金标检测试纸条。利用其对阴性参考样品、各型阳性参考样品、已知背景的田间样品进行检测。灵敏度试验结果显示:建立的试纸条方法敏感性高、可检测到病毒最低含量为0.98×104 LD50;特异性试验显示:在检测与口蹄疫临床症状相似病原———猪水泡病病毒(SVDV)、水泡性口炎(VS)、水泡性疹(VES)抗原时无交叉反应;重复性试验显示:不同批次间、同一批次内检测结果完全一致;符合性试验显示:检测国家口蹄疫参考实验室已确定口蹄疫病毒血清型的样品90份,阳性符合率、阴性符合率分别为96.70%、100%。本试验研发的口蹄疫病毒通用型金标检测试纸条具有很好的灵敏度、特异性、重复性和符合性,适合国内流行的口蹄疫病原的鉴别诊断。The aim of this study was to develop a sensitive, rapid and simple immunity colloid gold chromatographic analysis method for examination of foot-and-mouth disease, which can make the differential diagnosis with other infectious diseases. The purified anti-FMDV Asia 1 type coupling 12S antigen rabbit antibody were coupled with colloidal gold. The purified anti-FMDV A, O type coupling 12S antigen rabbit antibody and goat anti-rabbit IgG were wrapped onto nitrocellulose membrane as the test line （T line） and the control line （C line）, respectively. The FMDV general diagnosis kit was then performed. The seriate results indicated that sensitivity of the test kit reached 0. 98×104LD50. No cross reaction was found with SVD, VS, VES antigen by crosstests. In the clinic test, a total 90 field samples were detected with the test list. The result of corresponding rate for positive and negative samples were 96.70% and 100%. These experiments demonstrated that the established gold immunochromatographic strip test kit was sensitive, spe- cific for detecting FMDV and was potentially useful for first time and differential diagnosis.

关 键 词：口蹄疫病毒 偶联 通用型 金标检测试纸条 12S 

分 类 号：S852.659.6[农业科学—基础兽医学] S854.43[农业科学—兽医学]