為建立冷凍原腸中細(xì)菌基因組DNA的直接提取法，采用震蕩洗脫、金屬網(wǎng)過濾和梯度離心相結(jié)合的方法，對2份冷凍原腸樣品進(jìn)行前處理；對經(jīng)前處理后的原腸樣品，采用酚/氯仿法提取細(xì)菌基因組DNA，然后進(jìn)行瓊脂糖電泳和OD260/OD280比值測定；以細(xì)菌基因組DNA為模板，用PCR技術(shù)擴(kuò)增細(xì)菌16S rDNA并構(gòu)建克隆文庫，并從2個(gè)文庫中各隨機(jī)挑取3個(gè)菌落進(jìn)行16S rDNA 的PCR鑒定和DNA測序。結(jié)果顯示：樣品提取的DNA濃度、純度較高，OD260/OD280比值介于1.8~2.0；挑取的6個(gè)菌落中，1個(gè)為陰性，剩余5個(gè)為陽性，為腸球菌屬（Enterococcus）和魏斯氏菌屬（Weissella）的5種細(xì)菌。結(jié)果表明，本研究提取的原腸細(xì)菌基因組DNA質(zhì)量較好，可用于非培養(yǎng)法研究冷凍原腸中細(xì)菌的多樣性。本研究解決了因缺乏原腸細(xì)菌基因組DNA提取方法，無法進(jìn)一步應(yīng)用現(xiàn)代分子生物學(xué)方法研究冷凍原腸細(xì)菌的多樣性、菌群組成結(jié)構(gòu)和動態(tài)變化等難題。
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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