摘要
[目的]探索适宜的畜禽废水中活性污泥微生物总DNA提取的方法。[方法]采用2种方法提取畜禽废水中活性污泥微生物总DNA,即"提取缓冲液+蛋白酶K+SDS"提取法以及"溶菌酶-SDS-蛋白酶"提取法。通过对这2种方法进行比较,以确定最佳试验方案。[结果]紫外分光光度法分析表明,"提取缓冲液+蛋白酶K+SDS"提取法所得的OD260/OD280大于1.81。电泳结果显示,"提取缓冲液+蛋白酶K+SDS"提取法提取DNA亮度高,有大片断DNA的存在且拖带较少,条带集中,能成功进行16S rDNA的PCR扩增;通过PCR扩增所得的DNA的纯度检测结果显示,扩增出了条带分子约为1 500 bp的特异性条带"。溶菌酶-SDS-蛋白酶"提取法所提取的DNA没有得到较好的效果,点样孔较亮则可能存在一定的蛋白质残留,或其他杂质等,通过PCR扩增没有扩出特异性条带。[结论]"提取缓冲液+蛋白酶K+SDS"提取法为畜禽废水中微生物群落在分子水平上的研究提供了1种简便、可靠的DNA提取方法,为分析畜禽废水中细菌多样性提供了条件。
[Objective] The research aimed to find a method for DNA extraction from activated sludge. [Method] Two methods were chose to extract DNA from activated sludge. The one was ＂extraction buffer ＋ Proteinase K＋SDS＂, the other was ＂lysozyme -SDS- Proteinase＂. The best test project was confirmed by comparing the 2 methods. [Result] The results of ultraviolet spectro -photometric analysis showed that the combination of ＂extraction buffer ＋ Proteinase K ＋ SDS＂ was the most efficient, which OD260/ OD280 value of total DNA sample was 1.81. And the results of electrophoresis showed that the DNA strap was both bright and clear, which was fit for PCR amplification. The results of the purity detection on DNA got by PCR amplification showed that 1 specific band was amplified, which fragment size was about 1 500 bp. The effect of the combination of ＂lysozyme -SDS- Proteinase＂ was not good. There was no specific brand by amplification. [Conclusion] A rapid and credible method for direct extraction of DNA from activated sludge sample of livestock farm wastewater was found, which offered the condition for analyzing the variety of bacteria in livestock farm wastewater.
出处
《安徽农业科学》
CAS
北大核心
2008年第11期4452-4454,共3页
Journal of Anhui Agricultural Sciences
基金
四川农业大学青年科学创新基金(2006A057)
四川省公益性重大科技公关项目(2007NGY006)
关键词
畜禽废水
活性污泥
DNA提取
PCR扩增
Livestock farm wastewater
Activated sludge
DNA extraction
PCR amplification
