| Chelex-100法直接提取乳样中奶牛乳房炎主要致病菌DNA方法的建立 |
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| 引用本文： | 许金朋,郝永清.Chelex-100法直接提取乳样中奶牛乳房炎主要致病菌DNA方法的建立[J].中国畜牧兽医,2014,41(11):25-29. |
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| 作者姓名： | 许金朋 郝永清 |
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| 作者单位： | 内蒙古农业大学兽医学院微生物学与免疫学实验室, 内蒙古呼和浩特 010018 |
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| 基金项目： | 国家科技支撑计划(2012BAD12B09-02)"农牧交错区生态型现代奶业生产模式研究与示范";科技部成果转化项目(2012GB2A400058)"碘甘油乳头浸剂的中试生产及产业化开发"。 |
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| 摘 要： | 为建立一种直接从乳样中快速提取细菌DNA的方法,试验通过人工制备8个倍比稀释细菌的乳样,检测了Chelex-100法提取3种奶牛乳房炎主要致病菌(金黄色葡萄球菌、大肠杆菌和无乳链球菌)DNA进行PCR扩增的敏感性,并与苯酚—氯仿法进行了比较分析。结果显示,以Chelex-100法提取乳样中细菌DNA进行PCR扩增具有较高的敏感性,所检测金黄色葡萄球菌、大肠杆菌和无乳链球菌的最小浓度分别为103、102、102 CFU/mL;而使用苯酚—氯仿法提取乳样中各细菌DNA的PCR敏感性均为104 CFU/mL。综上所述,Chelex-100法提取乳样细菌DNA的PCR敏感性可以满足临床检测奶牛乳房炎的需要,显现了简单快速、经济、无污染的优点,为从乳样中直接提取细菌DNA提供了新的思路,对PCR快速检测乳房炎致病菌具有重要意义。
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| 关 键 词： | Chelex-100 奶牛乳房炎 细菌DNA PCR 敏感性 |
| 收稿时间： | 2014-04-30 |
Development of Chelex-100 as a Medium for Simple DNA Extraction of Major Mastitis Pathogens Directly from Milk |
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| XU Jin-peng,HAO Yong-qing.Development of Chelex-100 as a Medium for Simple DNA Extraction of Major Mastitis Pathogens Directly from Milk[J].China Animal Husbandry & Veterinary Medicine,2014,41(11):25-29. |
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| Authors: | XU Jin-peng HAO Yong-qing |
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| Institution: | Laboratory of Microbiology and Immunology, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China |
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| Abstract: | In this research,a simple and rapid extraction of microbial DNA for PCR using Chelex-100 chelating resin directly from milk was built.3 common mastitis pathogens (S.aureus,E.coli and S.agalactiae) were submitted to tenfold serial dilu-tions and artificially inoculated to milk samples.Then the sensitivities of the PCR assay using the Chelex-100 DNA extraction method were detected.In the meantime,the sensitivity of the 3 common mastitis using phenol-chloroform DNA extraction method was compared.The results showed that the PCR assay using the Chelex-100 DNA extraction method was more sensitive than that using phenol-chloroform DNA extraction method.The sensitivity of the PCR assay using the Chelex-100 DNA extraction method in detecting S.aureus, E.coli and S.agalactiae was 103,102 and 102 CFU/mL,repectively.The sensitivity of the PCR assay using phenol-chloroform DNA extraction method in detecting S.aureus,E.coli and S.agalactiae was all 104 CFU/mL.The thresholds of the sensitivities using the Chelex-100 DNA extraction method were compatible with their use as an efficient tool of diagnosis in bovine mastitis.The Chelex-100 DNA extraction method was simple,rapid,cheap,involve no organic solvents and meaningful for direct extraction of DNA of other pathogens from milk.Undoubtedly,this method will make sense for rapid diagnose of bovine mastitis by PCR. |
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| Keywords: | Chelex-100 bovine mastitis bacterial DNA PCR sensitivity |
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