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冷冻原肠中细菌基因组DNA提取方法的建立

6954 2020-03-12 12:06:05 宋鸿雁,仇保丰,高雪梅,等 中国动物检疫
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为建立冷冻原肠中细菌基因组DNA的直接提取法,采用震荡洗脱、金属网过滤和梯度离心相结合的方法,对2份冷冻原肠样品进行前处理;对经前处理后的原肠样品,采用酚/氯仿法提取细菌基因组DNA,然后进行琼脂糖电泳和OD260/OD280比值测定;以细菌基因组DNA为模板,用PCR技术扩增细菌16S rDNA并构建克隆文库,并从2个文库中各随机挑取3个菌落进行16S rDNA 的PCR鉴定和DNA测序。结果显示:样品提取的DNA浓度、纯度较高,OD260/OD280比值介于1.8~2.0;挑取的6个菌落中,1个为阴性,剩余5个为阳性,为肠球菌属(Enterococcus)和魏斯氏菌属(Weissella)的5种细菌。结果表明,本研究提取的原肠细菌基因组DNA质量较好,可用于非培养法研究冷冻原肠中细菌的多样性。本研究解决了因缺乏原肠细菌基因组DNA提取方法,无法进一步应用现代分子生物学方法研究冷冻原肠细菌的多样性、菌群组成结构和动态变化等难题。

Establishment of a Method for Extracting Bacterial Genome DNA from Frozen Archenterons 

In order to establish a direct method for extracting bacterial genome DNA from frozen archenterons,two samples of frozen archenterons were pretreated by combination of shake-elution,filtration via metal mesh and gradient centrifugation technique;then the bacterial genome DNA was extracted by use of phenol-chloroform for agarose gel electrophoresis and measurement of OD260/OD280 ratio;taking the bacterial genome DNA as the template,16S rDNA was amplified by PCR assay,then the clone libraries were constructed. Three bacterial colonies were randomly selected from each of the two libraries for PCR identification and DNA sequencing of 16S rDNA. The results showed that the concentration and purity of DNA extracted from the samples were relatively high,and the OD260/OD280 ratio ranged from 1.8 to 2.0;one out of six selected bacterial colonies was negative,and others were positive,furthermore,the five species of bacteria belonged to Enterococcus and Weissella. In conclusion,the bacterial genome DNA extracted from frozen archenterons was of good quality,which could be used to study the diversity of bacteria in frozen archenterons by culture-independent method. With the method established in this study,it was possible to conduct further study on bacterial diversity,community structure and variation by using modern molecular biology technologies.

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