爲建立冷凍原腸中細菌基因組DNA的直接提取法，采用震蕩洗脫了也、金屬網過(guò)濾和梯度離心相結合的方法，對(duì)又放2份冷凍原腸樣(yàng)品進(jìn)中得行前處理；對(duì)經(jīng)前處理後(hòu)的原腸樣(yàng)紅車品，采用酚/氯仿法提取細菌基因組DNA，然後(hòu火物)進(jìn)行瓊脂糖電泳和OD260/OD280比值測定；以細菌基因組DNA靜關爲模闆，用PCR技術擴增細菌16S rDNA海答并構建克隆文庫，并從2個文庫中各随機挑裡喝取3個菌落進(jìn)行16S rDNA 美哥的PCR鑒定和DNA測序。結果顯示：樣(yàng)品提取的DNA濃度、純度較高木新，OD260/OD280比值介于1.8~2.0；挑近校取的6個菌落中，1個爲陰性，剩餘5個爲陽性，爲腸球菌屬（En農哥terococcus）和魏斯氏菌屬（Weissella）的5種(zhǒng)匠化細菌。結果表明，本研究提取的原腸細菌基因組DNA質量較好(hǎo)，可用劇店于非培養法研究冷凍原腸中細菌的多樣(yàng)性。本研究解決了雪分因缺乏原腸細菌基因組DNA提取方法，無法進(jìn)一步應用你關現代分子生物學(xué)方法研究冷凍原腸細菌的多樣(yàng)性、菌群組成(c空遠héng)結構和動态變化等難題。
Establishment of 都我a Method for Extracting 作如Bacterial Genome DNA&nb裡鐘sp;from Frozen Archenterons
In order to establish a dir雜好ect method for extracting bacterial 務頻genome DNA from報這 frozen archenterons，two s音知amples of frozen archenteron人吃s were pretreated by combinati長子on of shake-elu到員tion，filtration via metal 美是mesh and gradient centri章工fugation technique；then the 樹業bacterial genome DNA was extracted 如數by use of phenol-chloroform for agaros件紅e gel electrophoresis and measurement來慢 of OD260/OD280 ratio；taking離用 the bacterial genome DNA as the tem件湖plate，16S rDNA was a東藍mplified by PCR assay，then湖民 the clone libr現就aries were constructed. Three 子林bacterial colonies were r木雪andomly selected from each of t呢靜he two libraries for PCR identificati劇紅on and DNA sequencing of 16S rDN老好A. The results showe花年d that the concentration an呢說d purity of DNA extracted from the samp來店les were relatively high，and t雪線he OD260/OD280 rati月可o ranged from 1.8 to 2.0；one o飛謝ut of six selected bacterial col水近onies was negative，男又and others were positive弟雨，furthermore，the five spec不中ies of bacteria belonged to Enterococcu南微s and Weissella. In conclusion，the b文做acterial genome 們和DNA extracted from fro就去zen archenterons was of good qua購樹lity，which could be used to study the 內山diversity of ba聽飛cteria in frozen archenteron自玩s by culture-inde制西pendent method. With th銀空e method established民舊 in this study，it was possible to c劇家onduct further study on bacterial dive數兒rsity，community structure and 了體variation by using modern 上火molecular biology te南花chnologies.
全文下載鏈接：https://kns.cnki.n山話et/KCMS/detail/detail.aspx家下?dbcode=CJFQ&dbname=CJFDAU能愛TO&filename=ZGDW202002020&v=MjY5M現公zJGeW5oVkw3T1B5clBlYkc0SE5ITXJZO森議UhaSVI4ZVgxTHV4WV音個M3RGgxVDNxVHJXTTFGckNVUjdxZlpPZHQ=
