奶牛乳房炎四种主要致病菌多重PCR检测方法的建立与应用

发布时间：2018-06-07 16:07

本文选题：奶牛乳房炎 + Chelex-100　；参考：《内蒙古农业大学》2015年硕士论文

【摘要】：奶牛乳房炎是影响中国乃至世界乳业发展的最重要的疾病之一,金黄色葡萄球菌、无乳链球菌、牛支原体和大肠杆菌等致病菌的感染是引起该病的最主要原因。本研究采用Chelex-100法从乳样中直接提取细菌DNA,分别检测了该法提取人工制备金黄色葡萄球菌、无乳链球菌、牛支原体或大肠杆菌乳样标本DNA进行PCR扩增的敏感性；选取金黄色葡萄球菌、无乳链球菌、牛支原体和大肠杆菌4种奶牛乳房炎主要致病菌的4对特异性引物,通过优化反应条件,建立可同时检测奶牛乳房炎4种主要致病菌的多重PCR方法；采用建立的多重PCR方法,检测临床采集的162份乳样,并与病原菌检测的“金标准”—培养法的结果进行比较。结果表明,Chelex-100法可经济、快速、有效地直接提取乳样细菌DNA,用于PCR检测奶牛乳房炎主要致病菌；建立的多重PCR方法可同时扩增奶牛乳房炎4种主要致病菌,具有良好的特异性和敏感性(检测无乳链球菌、牛支原体、大肠杆菌和金黄色葡萄球菌的最小浓度分别为1.25x103、6.25x102、6.25x102、 1.25x103 CFU/mL);多重PCR法与培养法检测临床采集乳样的结果表明,2种方法检测金黄色葡萄球菌、无乳链球菌和大肠杆菌3种细菌的结果基本一致,差异不显著(P0.05)；多重PCR法检测到的牛支原体乳样数多于培养法,差异极显著(P0.01)。本研究建立的乳样中奶牛乳房炎4种主要致病菌的多重PCR检测方法,可以满足临床经济、快速、有效地检测奶牛乳房炎4种主要致病菌的需要,对临床防控奶牛乳房炎具有重要的意义。
[Abstract]:Cow mastitis is one of the most important diseases affecting the development of dairy industry in China and the world. The infection of Staphylococcus aureus, Streptococcus acuminata, Mycoplasma bovis and Escherichia coli is the main cause of the disease. In this study, Chelex-100 method was used to directly extract bacterial DNA from milk samples. The sensitivity of PCR amplification of DNA from milk samples of staphylococcus aureus, streptococcus lactobacillus, Mycoplasma bovis or Escherichia coli was detected by this method. Four pairs of specific primers of Staphylococcus aureus, Streptococcus lactococcus, Mycoplasma bovis and Escherichia coli were selected to optimize the reaction conditions. A multiplex PCR method was established for simultaneous detection of four major pathogenic bacteria in dairy cow mastitis, and a multiplex PCR method was used to detect 162 milk samples collected in clinic, and the results were compared with the results of the "gold standard" -culture method for the detection of pathogenic bacteria. The results showed that Chelex-100 method could be used to detect the main pathogens of dairy mastitis by PCR, and to amplify the four main pathogens of mastitis simultaneously by using Chelex-100 method. Good specificity and sensitivity (detection of streptococcus actinis, Mycoplasma bovis, The minimum concentrations of Escherichia coli and Staphylococcus aureus were 1.25x103c6.25x102c6.25x102and 6.25x102respectively, 1.25x103 CFU / mL2.Multiplex PCR method and culture method were used to detect the milk samples of Staphylococcus aureus, and the results showed that the two methods were used to detect Staphylococcus aureus. The results of Streptococcus lactis and Escherichia coli were basically the same, the difference was not significant (P 0.05), and the number of milk samples of Mycoplasma bovis detected by multiple PCR method was more than that by culture method, and the difference was very significant. The multiplex PCR method for the detection of four main pathogens of dairy cow mastitis in milk samples established in this study can meet the needs of clinical economy, rapid and effective detection of four main pathogenic bacteria of dairy cow mastitis. It is of great significance for clinical prevention and control of cow mastitis.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23

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