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

Development of Chelex-100 as a Medium for Simple DNA Extraction of Major Mastitis Pathogens Directly from Milk

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。

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 dilutions 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 10^3,10^2 and 10^2 CFU/mL,repectively. The sensitivity of the PCR assay using phenol-chloroform DNA extraction method in detecting S. aureus s E, coli and S. agalactiae was all 10^4CFU/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.