本文選題：奶牛乳房炎 + Chelex-100��； 參考：《內(nèi)蒙古農(nóng)業(yè)大學》2015年碩士論文

【摘要】：奶牛乳房炎是影響中國乃至世界乳業(yè)發(fā)展的最重要的疾病之一,金黃色葡萄球菌、無乳鏈球菌、牛支原體和大腸桿菌等致病菌的感染是引起該病的最主要原因。本研究采用Chelex-100法從乳樣中直接提取細菌DNA,分別檢測了該法提取人工制備金黃色葡萄球菌、無乳鏈球菌、牛支原體或大腸桿菌乳樣標本DNA進行PCR擴增的敏感性；選取金黃色葡萄球菌、無乳鏈球菌、牛支原體和大腸桿菌4種奶牛乳房炎主要致病菌的4對特異性引物,通過優(yōu)化反應條件,建立可同時檢測奶牛乳房炎4種主要致病菌的多重PCR方法；采用建立的多重PCR方法,檢測臨床采集的162份乳樣,并與病原菌檢測的“金標準”—培養(yǎng)法的結果進行比較。結果表明,Chelex-100法可經(jīng)濟、快速、有效地直接提取乳樣細菌DNA,用于PCR檢測奶牛乳房炎主要致病菌；建立的多重PCR方法可同時擴增奶牛乳房炎4種主要致病菌,具有良好的特異性和敏感性(檢測無乳鏈球菌、牛支原體、大腸桿菌和金黃色葡萄球菌的最小濃度分別為1.25x103、6.25x102、6.25x102、 1.25x103 CFU/mL);多重PCR法與培養(yǎng)法檢測臨床采集乳樣的結果表明,2種方法檢測金黃色葡萄球菌、無乳鏈球菌和大腸桿菌3種細菌的結果基本一致,差異不顯著(P0.05)；多重PCR法檢測到的牛支原體乳樣數(shù)多于培養(yǎng)法,差異極顯著(P0.01)。本研究建立的乳樣中奶牛乳房炎4種主要致病菌的多重PCR檢測方法,可以滿足臨床經(jīng)濟、快速、有效地檢測奶牛乳房炎4種主要致病菌的需要,對臨床防控奶牛乳房炎具有重要的意義。
[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.
【學位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.23

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