微生物发酵床垫料微生物总DNA提取方法的研究

Extraction Methods for Bedding Microbial DNA from Fermentation Bed

传统的平板分离培养法,不能全面反映垫料微生物的基因信息,所以获得高质量的垫料微生物总DNA对于研究垫料微生物的群落结构就显得尤为重要。本实验设计对比了6种关于养猪发酵床垫料微生物总DNA的提取方法,对6种方法提取的DNA的浓度和纯度进行比较评价,结果表明,试剂盒法中因为没有专门针对本实验垫料的总DNA提取试剂盒,所采用的粪便,土壤以及淤泥总DNA提取试剂盒都没有达到很好的效果;而在化学法当中,单纯的SDS法和CTAB法都不能有效除去腐殖酸和杂蛋白的污染,通过核酸蛋白分析仪测定的A260/A280的比值基本都在1.5以下,说明纯度很低,用16S rDNA通用引物进行PCR扩增也没有得到目的条带。在SDS-CTAB结合法中利用10%的PEG-8000进行沉淀不进行回收纯化所提取的DNA具有完整性好,纯度高的优点,浓度达到200 ng.μL-1以上,通过核酸蛋白分析仪测定的A260/A280的比值达到1.8左右。用16S rDNA通用引物扩增得到比较亮的目的条带,因此SDS-CTAB结合法是一种高效、可靠的垫料微生物总DNA提取方法,有利于进行下游的分子生态学研究。

Traditional plate culture method can not fully reflect the microbial genetic information in microbiological fermentation bed,so obtaining high quality DNA is necessary to study microbial community structure in fermentation bed.In this research we designed and compared six extraction methods for bedding microbial DNA from fermentation bed,mainly in evaluating concentration and purification of extracted DNA.Results indicated that the kit methods have high pertinence,without specific bedding material microorganism DNA extraction kit,the use of excrement,soil and silt kit did not achieve good results.Simply applying SDS or CTAB methods could not effectively eliminate the pollution of humic acid and remove the complex proteins;so that,the ratio of A260 to A280 of the DNA was lower than 1.5 and no result obtained by PCR amplification.Comparatively,in SDS-CTAB method,using 10% PEG8000 in precipitation obtained the DNA with good integrity and high purity,the concentration of DNA was more than 200 ng·μL-1,the ratio of A260 to A280 was about 1.8,and the PCR amplified targeted the DNA.Results indicated that the SDS-CTAB was an efficient and reliable method for DNA extraction from microbiological fermentation bed and it would be also conducive to the research of molecular ecology.