污水处理系统中噬菌体的浓缩分离和宏合格基因组DNA的提取
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摘要:
以城市污水活性污泥为材料,经分级粗滤、超滤、等密度梯度离心等方法获得高纯度噬菌体悬液,常规SDS方法提取噬菌体DNA.荧光显微镜观察和计数表明浓缩液中噬菌体的纯度和丰度都显著提高;利用透射电镜对浓缩液进行观察,噬菌体的形态呈杆状、线状等多样;基因组提取结果显示,得到的DNA样品纯度高,大小介于20~25kb之间,电泳条带清晰,整个泳道没有弥散现象;利用细菌通用引物对提取的噬菌体DNA进行16SrDNA扩增阴性,说明所得到的噬菌体纯度较高,没有细菌污染.通过实验获得一种高效快捷的从污水处理系统中纯化浓缩噬菌体并进一步获得噬菌体宏基因组DNA的方法,同时为研究环境噬菌体生态分布、多样性组成奠定基础.
In this study, activated sludge from a municipal sewage treatment system were collected and used as samples. Bacteriophages suspension with high purity were obtained after a series of filters and elution, concentration, cesium chloride gradient centrifugation and dialysis, etc. Genomic DNA was extracted by using a Proteinase K and SDS method. Observation results under a fluorescence microscope indicated that the purity and abundance of phage suspensions were significantly in- creased after concentration. The purified bacteriophages were very diverse under a transmission electron microscopy including morphologies of rod, filiform, etc. The obtained bacteriophages genomic DNA each had a high purity that only one clear band was present on the electrophoresis gel with a size of 20--25 kb without any dispersion. The negative PCR detection results by using 16S rDNA universal primers confirmed that the obtained bacteriophages genomic DNA had no contamination by bacterial DNA. A quick and effective method is developed for separating and concentrating bacteriophages, purifying metagenomic bacte riophages DNA from activated sludge from a municipal wastewater treatment system by experiment. The obtained bacterio- phage samples can be further used for bacteriophage isolation, observation and cultivation. The obtained bacteriophage genomic DNA samples can be used for metagenomic analysis and bacteriophage composition and their ecological distribution study.
作者:
赵红丽 祁诗月 张斐 杨清香
机构地区:
betway官方app 生命科学学院 betway官方app 河南省高校资源微生物与功能分子重点实验室
出处:
《betway官方app 学报：自然科学版》 CAS 北大核心 2016年第4期131-136,共6页
基金:
国家自然科学基金(NSFC 21477035) 河南省杰出创新人才基金资助(144200510007)
关键词:
噬菌体 浓缩 宏基因组DNA 活性污泥
bacteriophages concentration metagenomic DNA activated sludge
分类号:
Q939.9 [生物学—微生物学]
