猪源大肠杆菌Nissle 1917菌株的分离鉴定

发布时间：2018-04-27 10:30

  本文选题：大肠杆菌Nissle + 分离鉴定　； 参考：《微生物学通报》2017年04期

【摘要】：【目的】证实大肠杆菌Nissle 1917作为自然菌株存在于动物猪体内,并能从猪粪便中分离。建立大肠杆菌Nissle 1917的原位杂交鉴定方法。【方法】采集135份健康断奶仔猪的新鲜粪便制备DNA模板,以人源大肠杆菌Nissle 1917为阳性对照菌株,分别针对Nissle 1917的I型菌毛亚单位Fim A、F1C菌毛亚单位Foc A及两个质粒pMUT1和pMUT2的相关基因序列设计5对特异性引物进行PCR扩增;并将其中427 bp大小的质粒片段pMUT2(a)作为目的片段回收纯化,用地高辛随机引物标记法制成DNA探针。【结果】从其中的2份DNA模板中扩增出上述5对特异性引物PCR预期大小相符的片段,初步认为大肠杆菌Nissle 1917可能存在于猪体内。应用制备的探针通过菌落原位杂交的方法从2份阳性粪便样品中筛选出2株阳性菌落,通过血清学检验、PCR扩增和测序进一步鉴定为阳性Nissle 1917菌株。【结论】动物源益生菌Nissle 1917的分离鉴定,为优良动物源益生菌研究和应用奠定了基础。
[Abstract]:[objective] to confirm that Escherichia coli Nissle 1917 exists as a natural strain in animal pigs and can be isolated from pig feces. An in situ hybridization method for identification of Escherichia coli Nissle 1917 was established. [methods] DNA template was prepared from fresh feces of 135 healthy weaned piglets. Nissle 1917 was used as positive control strain. Five pairs of specific primers were designed for PCR amplification of Nissle 1917 type I pili subunit Foc A and two plasmids pMUT1 and pMUT2 related genes. The plasmid fragment of 427 BP in size was recovered and purified as the target fragment. Digoxigenin random primer labeling method was used to make DNA probe. [results] five pairs of specific primer PCR fragments were amplified from the two DNA templates. It was suggested that E. coli Nissle 1917 might exist in pigs. Two positive colonies were screened from 2 fecal samples by colony in situ hybridization. The positive strain of Nissle 1917 was further identified by serological analysis and sequencing. [conclusion] the isolation and identification of probiotic strain Nissle 1917 from animal sources laid a foundation for the study and application of excellent probiotics from animals.
【作者单位】： 扬州大学兽医学院;江苏省动物重要疫病与人兽共患病防控协同创新中心;国药集团扬州威克生物工程有限公司;
【基金】：国家自然科学基金项目(No.31672579,30571374,30771603,31072136,31270171) 江苏高校优势学科建设工程资助项目 科技部转基因生物新品种培育重大专项(No.2014ZX08006-001B)~~
【分类号】：S852.61
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本文编号：1810366