奶牛乳房炎主要致病菌16S rDNA基因芯片检测方法的建立  

Detection of four bacterial species in bovine mastitis samples by DNA microarray based on the 16S rDNA sequences

作　　者：邢会杰[1,2] 曹荣峰[3] 师志海[1] 贾坤[1] 杨林[4] 林志雄 李守军[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]暨南大学实验动物中心,广东广州510642 [3]青岛农业大学动物科技学院,山东青岛266109 [4]中国动物疫病预防控制中心,北京100026 [5]广东进出口检验检疫局,广东广州510642

出　　处：《中国预防兽医学报》2008年第12期939-943,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：广东省科技攻关计划(2005B20201004、2007A020100006-2);教育部“长江学者和创新团队发展计划”创新团队项目(IRT0723)

摘　　要：本研究建立了快速检测奶牛乳房炎主要致病菌的基因芯片方法,试验以奶牛乳房炎4种主要致病菌的16S rDNA基因作为基因芯片检测的靶片段,设计和筛选通用性引物和特异性探针,对通用引物进行荧光标记、PCR扩增、芯片杂交和信号扫描分析,根据杂交信号强度和聚类分析结果,判定奶牛乳房炎感染的致病菌种类。实验结果显示:以16S rDNA为靶基因检测奶牛乳房炎主要致病菌(无乳链球菌、肺炎克雷伯氏菌、奇异变形杆菌、金黄色葡萄球菌)的基因芯片方法,可以快速、特异、准确地对这4种试验菌株进行检测和鉴定。该检测方法的建立将为流行病学调查、食品卫生监督、乳房炎的防控等提供快速、有效的检测方法,具有良好的市场应用前景。Conventional methods for detection of different bacterial species causing bovine mastitis have been time cusuming and less reliable. A rapid, sensitive, specific diagnostic test to identify and diagnose mastitis pathogens would be valuable for selecting reasonable therapeutic regimen. In this study, DNA microarray method was established for detecting 4 stains of mastitis pathogenic bacteria using 16S rDNA gene as the target. A universal PCR primer pair was designed based on the target pathogen＇s 16S rDNA sequence, labeled with fluorescent Cy3, and hybridized with the PCR products printed on the slide. Four strains of bovine mastitis were successfully discriminated based on the hybridization signal and cluster analysis results. The sensitivity of detection was approximately 103 CFU/mL. These results supported the application of DNA microarray method in concurrent detection of bacterial species causing bovine mastitis.

关 键 词：基因芯片 奶牛乳房炎 致病菌 聚合酶联反应 

分 类 号：Q811.4[生物学—生物工程]