改良DNA染色法对支原体检测的效果评估  

Effect evaluation of improved DNA staining method for Mycoplasma detection

作　　者：王晓燕 刘军 于佳 王凯 房世娣 魏然 冯德杰 WANG Xiao-yan;LIU Jun;YU Jia;WANG Kai;FANG Shi-di;WEI Ran;FENG De-jie(The Quality Control Department,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司质量检定室甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2022年第4期31-36,共6页Progress In Microbiology and Immunology

摘　　要：目的评估改良的DNA染色法检测支原体的效果。方法使用无水乙醇为固定液和DAPI为荧光染料进行改良的DNA染色法,通过改变细胞接种浓度、细胞培养时间、接种支原体的浓度以及接种猪鼻支原体、口腔支原体和精氨酸支原体,观察支原体污染细胞进行DNA染色后的效果变化。结果Vero细胞接种浓度在1.0×10^(5)个/mL时,DNA染色效果最好;支原体在传代培养5 d后进行DNA染色效果更好;接种支原体污染的细胞上清,胞核外有较少的蓝色荧光,而接种支原体污染的细胞培养物的胞核外可见大量的蓝色荧光;猪鼻支原体的DNA染色效果比口腔支原体和精氨酸支原体的DNA染色效果好。结论改变细胞接种浓度、细胞培养时间、支原体浓度以及种类,都能观察到不同的DNA染色结果,从而证明改良的DNA染色法也能用于支原体DNA染色检查。Objective To evaluate the effect of improved DNA staining method in detection of Mycoplasma.Methods The improved DNA staining method using anhydrous ethanol as fixative and DAPI as fluorescent dye was applied to observe the effect of DNA staining results by changing cell inoculation concentration,cell culture time,Mycoplasma concentrations and species(Mycoplasma hyorhinis,Mycoplasma orale and Mycoplasma arginini).Results The optimal DNA staining result was obtained when the inoculation concentration of Vero cells was 1.0×10^(5)cells/mL and culture time after cell passage was 5 d.There was less blue fluorescence in cells inoculated with the supernatants of Mycoplasma hyorhinis-infected Vero cells,compared to the infected cells continually cultured after passage.The staining effect of Mycoplasm hyorhinis-infected cells was better than that of Mycoplasm orale-and Mycoplasm arginini-infected cells.Conclusion The improved DNA staining method is also applicable to Mycoplasma detection owing to the positive staining results proved in different conditions including cell inoculation concentrations,culture time,Mycoplasma species.and their concentrations.

关 键 词：DNA染色法 支原体 VERO细胞 细胞培养 DAPI 

分 类 号：R378[医药卫生—病原生物学]