【摘要】 为建立快速并能同时检测无乳链球菌、金黄色葡萄球菌、绿脓杆菌3种奶牛隐性乳房炎致病菌的方法,根据无乳链球菌16S r RNA基因、金黄色葡萄球菌NUC耐热基因、绿脓杆菌ETA基因序列并参考文献各合成一对特异性引物,通过对PCR反应各项参数的调整优化,建立多重PCR检测体系,并同时进行灵敏性试验和特异性试验。结果显示:该检测方法可以同时扩增出无乳链球菌270 bp、金黄色葡萄球菌153 bp和绿脓杆菌375 bp的特异性片段以及细菌16S rRNA 1500 bp的通用片段,而对于其他的6种病原菌只能扩增出1 500 bp的通用片段,具有较强的特异性。三种特异性引物扩增条带与Gen Bank上的细菌基因序列同源性为99%。增菌培养24h,该体系对无乳链球菌、金黄色葡萄球菌、绿脓杆菌的最低灵敏度分别为8×10⁴、4×10⁴、1. 5×10⁵CFU·m L^-1。本研究建立的检测方法能够完成无乳链球菌、金黄色葡萄球菌、绿脓杆菌的快速检测,对于生产上奶牛隐性乳房炎的鉴别诊断和快速检测具有重大意义。更多还原

【Abstract】 In order to establish a method for the rapid and simultaneous detection of three pathogenic bacteria of mastitis of Streptococcus agalactiae,Staphylococcus aureus and Pseudomonas aeruginosa,three pairs of specific primers were synthesized based on the Streptococcus agalactiae 16 S rRNA gene,Staphylococcus aureus NUC heat-resistance gene,Pseudomonas aeruginosa ETA gene sequence and references. By adjusting and optimizing the parameters of the PCR reaction,a multiplex PCR detection system was established,and the sensitivity test and specificity test were simultaneously performed. The results showed that the detection method can simultaneously amplify specific fragments of 270 bp of Streptococcus agalactiae,153 bp of Staphylococcus aureus and 375 bp of Pseudomonas aeruginosa and a generic 1 500 bp fragment of bacterial 16 S rRNA. For other six kinds of pathogenic bacteria,only 1 500 bp universal fragments could be amplified,which had strong specificity. Three specific primer amplification bands were 99% homologous to the Gen Bank and bacterial gene sequences. After culture for 24 h,the minimum sensitivity of the system to Streptococcus agalactiae,Staphylococcus aureus and Pseudomonas aeruginosa was 8 × 10⁴,4 × 10⁴ and 1. 5 × 10⁵ CFU·m L^-1,respectively. The detection method established in this study can complete the rapid detection of Streptococcus agalactiae,Staphylococcus aureus and Pseudomonas aeruginosa,and is of great significance for the differential diagnosis and rapid detection of subclinical mastitis in dairy cows.更多还原