化脓隐秘杆菌间接免疫荧光方法的建立及应用  

Set-up a indirect immunofluorescence assay and its application for Trueperella pyogenes diagnosis

作　　者：刘威[1,2] 何深宏 任绍科 李小燕 杨雪芬[1] 程方俊 周作勇 曹立亭[1,2] LIU Wei;HE Shenhong;REN Shaoke;LI Xiaoyan;YANG Xuefen;CHENG Fangjun;ZHOU Zuoyong;CAO Liting(College of Animal Science,Southwest University,Chongqing 402460,China;Veterinary Science Engineering Research Center of Chongqing,Chongqing 402460,China)

机构地区：[1]西南大学动物科学学院,重庆402460 [2]重庆市兽医科学工程研究中心,重庆402460

出　　处：《湖南农业大学学报（自然科学版）》2020年第2期230-235,共6页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：国家重点研发计划项目(2018YFD0501705);“十二五”国家科技支撑计划项目(2011BAD36B00);重庆市留学人员回国创业创新支持计划项目(CX2017103)。

摘　　要：通过制备兔抗化脓隐秘杆菌(Trueperella pyogenes)高免血清,筛选固定方法、封闭液、洗涤液、抗体稀释度、抗体孵育时间等应用条件,建立一种检测化脓隐秘杆菌的间接免疫荧光方法。结果表明:用冷丙酮作固定剂,10%山羊血清作封闭液,0.05%Tween–20的0.01 mol/L pH 7.4 PBS(磷酸缓冲盐溶液)作洗涤液,兔抗化脓隐秘杆菌高免血清(一抗)1∶50稀释,37℃作用2 h,羊抗兔FITC(二抗)1∶100稀释,37℃作用2 h,草酸铵结晶紫衬染菌体3 min,对纯培养物化脓隐秘杆菌检测,可见清晰的特异性荧光,能区分溶血隐秘杆菌、伪结核棒状杆菌、金黄色葡萄球菌、大肠埃希菌、沙门氏菌、猪链球菌;在人工感染化脓隐秘杆菌死亡小鼠的心脏、肝脏、脾脏、肺脏、肾脏及发病山羊临床病料中均能检出化脓隐秘杆菌。An indirect immunofluorescence assay(IFA)was set up to detect Trueperella pyogenes using the rabbit hyperimmune serum. Specific conditions including fixing strategy, blocking solution, washing solution, antibody dilution, and antibody incubation time had been tested. The results showed that cold acetone as the fixative, 10% goat serum as the blocking solution, and 0.05% Tween-20 of 0.01 mol/L pH7.4 PBS as the washing solution were optimal for the method. And, the rabbit anti-Trueperella pyogenes hyperimmune serum with dilution 1∶50, incubation for 2 h at 37 ℃, and secondary antibody with dilution 1∶100, incubation for 2 h at 37 ℃, by use of ammonium oxalate crystal violet as stain for 3 min, the specific fluorescence was clearly visible in the pure cultivation of Trueperella pyogenes. It can clearly distinguish Arcanobacterium hemolysis, Corynebacterium pseudotuberculosis, Staphylococcus aureus, Escherichia coli, Salmonella, Streptococcus suis. To verify the method, in dead mouse tissues infected Trueperella pyogenes including heart, liver, spleen, lungs and kidneys, and diseased goats clinical sample, Trueperella pyogenes were detected.

关 键 词：山羊 小鼠 化脓隐秘杆菌 高免血清 间接免疫荧光方法 特异性 灵敏度 

分 类 号：S852.612[农业科学—基础兽医学]