奶水牛乳房炎致病菌的分离鉴定及多重PCR检测方法的建立

发布时间：2018-06-15 18:01

本文选题：水牛乳房炎 + 分离鉴定　；参考：《广西大学》2015年硕士论文

【摘要】：本研究对广西奶水牛88份乳样进行病原菌分离鉴定,根据引起奶水牛乳房炎的主要致病菌种类建立多重PCR快速诊断方法,并对该检测方法进行优化和临床检测评价。实验内容:1、通过对88份奶样进行细菌分离鉴定,从56份奶样中分离出112株病原菌,包括葡萄球菌47株,占41.9%;链球菌25株,占22.3%;肠杆菌21株,占18.8%;真菌12株,占10.8%;其它细菌7株,占6.2%。2、根据引起水牛乳房炎主要致病菌金黄色葡萄球菌nuc基因、大肠杆菌rrnB基因,无乳链球菌ef-tu基因,表皮葡萄球菌se2313基因序列设计选择4对特异性引物,建立并优化同时检测这4种病原菌的4重PCR快速诊断方法。实验结果表明:1、引起水牛乳房炎的主要致病菌为金黄色葡萄球菌、表皮葡萄球菌、无乳链球菌和大肠杆菌,还有部分真菌感染;感染主要以混合感染为主,56份感染奶样中有40个为2种及以上的多种致病菌混合感染,占71.4%。2、PCR产物进行电泳,大肠杆菌、金黄色葡萄球菌、表皮葡萄球菌、无乳链球菌分别在722bp、279bp、581bp和197bp处扩增出相应目的条带,且无其它非特异性产物,实验与临床检测结果证明该方法敏感性好,特异性高,与传统细菌分离检测方法相比,既保证结果准确,又大幅度缩短检测时间,提高检测效率。
[Abstract]:In this study, 88 milk samples of Guangxi milk buffalo were isolated and identified. The rapid multiplex PCR method was established according to the main pathogenic bacteria causing mastitis of milk buffalo, and the detection method was optimized and evaluated clinically. Experiment content: 1, through the isolation and identification of bacteria from 88 milk samples, 112 strains of pathogenic bacteria were isolated from 56 milk samples, including 47 strains of staphylococcus, accounting for 41.9%; 25 strains of streptococcus, 22.3x; 21 strains of Enterobacter, 18.8%; 12 strains of fungi, 10.8%; and 7 strains of other bacteria, According to the sequence of Staphylococcus aureus nuc gene, Escherichia coli rrnB gene, ef-tu gene and se2313 gene of Staphylococcus epidermidis, 4 pairs of specific primers were designed. To establish and optimize a 4-fold PCR rapid diagnostic method for simultaneous detection of these four pathogens. The results showed that the main pathogens causing buffalo mastitis were Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus lactococcus and Escherichia coli, and some fungal infections. 40 of 56 milk samples were mixed with two or more kinds of pathogenic bacteria, accounting for 71.4% of PCR products, E. coli, Staphylococcus aureus, Staphylococcus epidermidis, E. coli, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis. The corresponding target bands were amplified from 722bpn279bp581 BP and 197bp, respectively, and there were no other nonspecific products. The experimental and clinical results showed that this method was more sensitive and specific than the traditional bacterial isolation and detection methods. It not only ensures the accuracy of the results, but also greatly shortens the detection time and improves the detection efficiency.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23

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