【摘要】 脆弱拟杆菌作为严格厌氧菌,对培养环境要求苛刻,分离培养有较大难度。试验通过改进的选择性培养结合特异引物方法分离兔源脆弱拟杆菌,并利用16srDNA测序对其进行鉴定,为后续研究其与宿主关系奠定基础。试验从健康青年家兔肠道中采集内容物,先接种于含液态选择性培养基的厌氧管中,进行富集培养1~2d,然后将培养的菌液稀释后接种到选择性培养基平板上,于厌氧罐中培养1~3d,待长出菌落后,观察菌落形态,挑选符合脆弱拟杆菌菌落特点的单一菌落,培养后提取DNA,以脆弱拟杆菌特异引物进行PCR检测。对扩增出特异条带的菌株,扩增其16srDNA序列,并进行测序,测序结果与GenBank中脆弱拟杆菌序列进行比对,以鉴定分离的菌株。结果表明,细菌为大小不一的杆菌,革兰氏染色呈阴性。特异引物PCR产物电泳检测显示,挑取的27株单一菌落中,有7株可以扩增出符合目的片段大小的产物。随机选取两株进行16srDNA测序,其序列与GenBank中的递交的脆弱拟杆菌序列相似度大于99%,与其他细菌相似度均小于93.43%,可确定分离到的菌株为脆弱拟杆菌。更多还原

【Abstract】 As a strictly anaerobic acterium,Bacteroides fragilis was difficult to isolate and cultivate because of the strict growing condition.This experiment was conducted to isolate and identify rabbit original Bacteroides fragilis by combining specific culture medium and molecular techniques.The isolated rabbit original Bacteroides fragilis will provide the material to further research on the relationships between bacteria and its host.The intestinal content was collected from healthy young rabbit and inoculated to the liquid selective medium in anaerobic tube,and cultured for 1-2days.Subsequently,the cultured bacteria were inoculated to the plate culture medium and cultured for 1-3days in a simple anaerobic jar.After colonies were formed,27 colonies were picked out from it,and then one part of each colony was used for preservation and the remaining was cultured to extract genomic DNA for PCR detection using Bacteroides fragilis specific primers.Finally,16 srDNA sequences of the PCR-positive colonies were cloned using universal primers and sequenced.There were 7strains with Bacteroides fragilis specific bands when detecting the PCR products.Aligning the 16 srDNA sequence of these strains with the database in GenBank showed that it has more than 99%similarity with the 16 srDNA sequence of Bacteroides fragilis in GenBank and the highest similarity with other bacteria is 93.43%.更多还原