乳房炎3种病原菌DNA提取方法比较研究

Comparison of DNA Extraction Methods of Major Mastitis-causing Bacteria in Goat Milk

旨在比较3种DNA提取方法对山羊乳中致乳房炎主要病原菌DNA的提取效果及对PCR检测的影响。将不同浓度的金黄色葡萄球菌、无乳链球菌和大肠埃希氏菌加入羊乳中,并分别用硅胶膜试剂盒、酚/氯仿法和CTAB/NaCl法等3种方法提取总DNA,比较所获得DNA的浓度、纯度、所需时间和经济成本及对各病原特异性PCR检测的影响。结果显示,各方法均能成功地提取羊乳中致乳房炎的3种病原菌DNA;硅胶膜试剂盒和CTAB/NaCl法提取山羊乳中金黄色葡萄球菌和无乳链球菌DNA的效果相同且明显优于酚/氯仿法;3种方法提取山羊乳中大肠埃希氏菌的效果相同。CTAB/NaCl法具有提取效果好、耗时较短、成本低和方便的优点,是从羊乳中提取病原DNA的良好方法。研究结果为临床上山羊乳房炎病原菌的分子检测和流行病学调查提供了方法学上的参考。

The objective of this study was to evaluate the performance of three DNA extraction methods for recovery of the main mastitis-causing bacteria from goat milk and their effect on PCR detection.Different concentrations of bacteria were added to the goat milk,and the total DNA was extracted by 3 different methods:commercial DNA extraction kit,phenol/chloroform assay and CTAB/NaCl method.The concentration,purity of extracted DNA,as well as time and economical cost of different methods were compared,and the performance of three methods on following PCR detection was evaluated intensively.The results showed that,while all methods could effectively extract bacterial DNA form the goat milk,commercial DNA extraction kit and CTAB/NaCl assay exhibited better performance than phenol/chloroform method to obtain Streptococcus agalactiae and Staphylococcus aureus DNA.All 3 methods showed same performance on extraction of Escherichia coli DNA from goat milk.The overall evaluation of three methods suggested that CTAB/NaCl method has the advantages of low cost,good DNA extraction efficiency,convenience,and therefore is recommended for bacterial DNA extraction form goat milk.The results obtained in this study provided a methodological reference for molecular detection and epidemiological investigation.