间接免疫荧光显微术检测泡桐丛枝病原MLO的研究

Detection of Mycoplasma-like Organism in the Infected Paulownia by Indirect Immunofluorescence Microscopy

摘要: 以感染类菌原体MLO的泡桐组培苗为病原繁殖材料,采用差速离心和Percoll不连续密度梯度离心来提纯MLO作为免疫抗原制备兔抗血清。抗血清经健康汁液吸收后,作为第一抗体,用异硫代氰酸荧光素FIFC标记的羊抗兔免疫球蛋白作为第二抗体,进行间接免疫荧光显微镜检查染病组织中的MLO特异性荧光。经与DAPI染色技术比较,找出了适宜的徒手切片和减少非特异性反应的途径。此技术具有灵敏度高、特异性强和测定简便等特点。适用于MLO组织定位、组培苗脱毒效果评估、病原株系鉴定和病害检疫等。

Abstract: Using paulownia witches’broom MLO extracted and concerntrated from infected paulownia tissue culture plantlets by differential and discontinuous Percoll density centrifugation as antigen,the antiserum was produced.After the absorption with healthy paulownia extract,the antiserum could effectively detect paulownia witches’broom MLO in different tissues of infected plants with FITC Iabelled sheep-anti-rabbit IgG by indirect immunofluoresence microscopy. Compared with DAPI staining test,a more specific,sensitive and handy method for detecting MLO was developed,which would facilitate the detection of MLO in situ,identification of MLO strains and the diagnosis of plant diseases associated with MLOs.