通用基因芯片对奶牛乳房炎主要致病茵的检测

应用生物信息学手段和查阅文献资料设计了金黄色葡萄球菌、大肠埃希菌、无乳链球菌、绿脓杆菌、停乳链球菌、乳房链球菌6种奶牛乳房炎主要致病菌的通用引物和金黄色葡萄球菌、大肠埃希菌、绿脓杆菌的寡核苷酸探针及无乳链球菌、停乳链球菌、乳房链球菌的特异引物,并用这3种特异引物扩增片段的纯化产物作为这3种链球菌的检测探针。在引物对样品中细菌的相应基因片段扩增的同时进行靶基因的生物素标记,扩增的产物与硝酸纤维素膜上的探针进行杂交,酶联、显色后根据芯片扫描仪的判读结果来确定奶牛乳房炎致病菌感染的种类。结果表明,建立的以16S rDNA为对象的基因芯片技术可以快速的检测出以上6种细菌,整个检测过程需要6h~7h,灵敏度高,特异性好,能快速的对奶牛乳房炎的主要致病菌做出诊断。

Uiversal Gene Chip for Detection of the Pathogenic Bacteria of Cow Mastitis

Using bioinformatics measure and exploring materials, we designed universal primers of six species of pathogenic bacteria: E.coli, P.aeruginosa, S.aureus, S.agalactiae, S.dysgalactiae and S.uberis and oligo probes of E.coli, P.aeruginosa and S.aureus. At the same time special primers of S.agalactiae, S.dysgalactiae and S.uberis were also designed. Purified product which was amplified by three sorts of special primers became detection probes of three species of Streptococcus. while corresponding genes of bacteria were amplified, corresponding gene was biotin-labeled. Amplified products were hybridized with probes. According to the recorded signals,the species of pathogenic bacteria were determined. Results showed that the detection system which employed 16S rDNA can identify above six species of pathogenic bacteria, hybridization and data acquisition lasted about 6~7 hours, the sensitivity was improved greatly and specificity was high.