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

›› 2014, Vol. 41 ›› Issue (11): 25-29.

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

许金朋, 郝永清

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

收稿日期:2014-04-30 出版日期:2014-11-20 发布日期:2014-12-06
通讯作者: 郝永清 E-mail:haoyq1960@163.com
作者简介:许金朋(1990-),男,河北人,硕士生,研究方向:预防兽医学。
基金资助:
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"。

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

XU Jin-peng, HAO Yong-qing

Laboratory of Microbiology and Immunology, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China

Received:2014-04-30 Online:2014-11-20 Published:2014-12-06

摘要/Abstract

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。

关键词: Chelex-100; 奶牛乳房炎; 细菌DNA; PCR; 敏感性

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 10⁴CFU/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.

Key words: Chelex-100; bovine mastitis; bacterial DNA; PCR; sensitivity

中图分类号:

S858.23

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]. , 2014, 41(11): 25-29.

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.

参考文献

1 沙莎,宋振辉.动物微生物实验教程[M].重庆:西南师范大学出版社,2011.
2 周月琴,朱伟,刘志萍,等.用Chelex-100快速提取微量血痕中的DNA[J].复旦学报(医学版),2003,30(4):379～380.
3 赵秀玲,闻伟刚,余旭平,等.Chelex-100快速提取动物源饲料DNA方法的建立[J].上海交通大学学报(农业科学版),2004,22(1):82～85.
4 韩旭,武瑞,孙东波.奶牛乳房炎主要病原菌基因检测技术研究进展[J].中国畜牧兽医,2012,39(3):230～232.
5 Amin A S,Hamouda R H.PCR assays for detecting major pathogens of mastitis in milk samples[J].World Journal of Dairy & Food Science,2011,6(2):199～206.
6 Bramley A J,Dodd F H.Reviews of the progress of dairy science:Mastitis control-progress and prospects[J].Journal of Dairy Research,1984,51(3):481～512.
7 Cremonesi P,Castiglioni B,Malferrari G,et al.Technical note:Improved method for rapid DNA extraction of mastitis pathogens directly from milk[J].Journal of Dairy Science,2006,89(1):163～169.
8 Cressier B,Bissonnette N.Assessment of an extraction protocol to detect the major mastitis-causing pathogens in bovine milk[J].Journal of Dairy Research,2011,94(5):2171～2184.
9 Lamballerie X,Zandotti C,Vignoli C,et al.A one-step microbial DNA extraction method using Chelex 100 suitable for gene amplification[J].Microbiological Research,1992,143(8):785～790.
10 Martinez G,Harel J,Gottschalk M.Specific detection by PCR of Streptococcus agalactiae in milk [J].Canadian Journal of Veterinary Research,2001,65(1):68～72.
11 Phuektes P,Mansell P D,Browning G F.Multiplex polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and Streptococcal causes of bovine mastitis[J].Journal of Dairy Science,2001,84(5):1140～1148.
12 Riffon R,Sayasith K,Khalil H,et al.Development of a rapid and sensitive test for identification of major pathogens in bovine mastitis by PCR[J].Journal of Clinical Microbiology,2001,39(7):2584～2589.
13 Tamarapu S,Mckillip J L,Drake M.Development of a multiplex polymerase chain reaction assay for detection and differentiation of Staphylococcus aureus in dairy products[J].Journal of Food Protection,2001,64(5):664～668.
14 Walsh P S,Metzger D A,Higushi R.Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material[J].Bio Techniques,2013,54(3):134～139.

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