奶牛乳房炎链球菌的刃天青微量板快速诊断与药敏试验  

The Resazurin Microplate Assay for Quick Diagnosis and Antibiotic Sensitivity Determination of Bovine Mastitis-causing Streptococci

作　　者：李洋洋[1] 李光亚[1] 包利平[1] 史蕾[1] 王娟[1] 孙怀昌[1] 

机构地区：[1]扬州大学兽医学院,江苏省重要动物疫病与人兽共患病协同创新中心,扬州225009

出　　处：《中国奶牛》2016年第1期26-31,共6页China Dairy Cattle

基　　金：江苏高校优势学科建设工程资助项目(PAPD)

摘　　要：本研究以刃天青钠取代链球菌选择培养基EN的溴甲酚紫指示剂,建立了奶牛乳房炎链球菌快速诊断与药敏试验用微量板。将不含指示剂EN的培养基分装到96孔板,接种不同细菌或其感染模拟奶样,培养2h加入优化浓度的刃天青指示剂,结果表明能显示无乳链球菌、停乳链球菌和乳房链球菌生长,不能显示大肠杆菌和葡萄球菌生长;检测三种奶牛乳房炎链球菌总敏感性达105cfu/m L,能在10h内获得诊断结果。60份临床奶样诊断结果显示,刃天青微量板法检测链球菌阴性奶样特异性达100%,检测链球菌单阳性奶样敏感性为100%,检测链球菌混合阳性奶样敏感性达97.1%。用矫正浓度的10种抗生素包被微量板,分别用10株链球菌纯培养、感染模拟奶样和9份链球菌阳性奶样进行刃天青微量板法快速药敏试验,所获结果与标准纸片扩散法完全一致。试验结果表明,本研究建立的刃天青微量板法可取代标准方法用于奶牛乳房炎链球菌的快速诊断及药敏试验。To establish a microplate assay for quick diagnosis and antibiotic sensitivity determination of bovine mastitis-causing streptococci, the bromocresol purple indicator in streptococcal selective medium EN was replaced with an optimized concentration of resazurin. The EN medium without indicator was aliquoted onto 96-well microplates and different bacteria or their infection mimicking milk samples were inoculated. After cultivation for 2h, 0.04% resazurin was added and the cultivation was continued for an additional 12h. The bacterial growth was determined according to the medium color change from dark blue to purplish red. The results showed that the resazurin microplate could indicate the growth of Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis, but not Staphylococcus. aureus and E.coli. The conclusive diagnosis could be obtained within 10h with an overall detection sensitivity of 105CFU/mL streptococci. The testing results of 60 milk samples showed that the resazurin microplate assay had a sensitivity of 100% for detecting streptococcus-positive milk samples or 97.1% for detecting mixed bacterial milk samples with a specificity of 100%. Ten different antibiotics at rectified concentrations were coated onto microplates and the quick antibiotic sensitivity assay was performed using 10 reference streptococcal strains and 9 streptococcus-positive milk samples. The results showed that the resazurin microplate antibiotic sensitivity assay had a 100% agreement with the standard disc diffusion method. These data suggest that the resazurin microplate assay could replace the standard methods for quick diagnosis and antibiotic sensitivity determination of bovine mastitis-causing streptococci.

关 键 词：奶牛乳房炎 链球菌 快速诊断 刃天青微量板法 快速药敏试验 

分 类 号：S858.23[农业科学—临床兽医学]