猪源大肠杆菌Nissle 1917菌株的分离鉴定

Isolation and identification of porcine-originated Escherichia coli Nissle 1917

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FimA、F1C菌毛亚单位FocA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 verify that Escherichia coli Nissle 1917 also exists in swine as a natural isolate and could be isolated from swine faeces. The method for identifying the Escherichia coli Nissle 1917 using in situ hybridization was established. [Methods] All 135 pieces of fresh faeces from healthy weaned piglets were collected to prepare DNA template samples. And the DNA template samples were screened and tested by PCR with five pairs of specific primers targeting the chromosomal genes encoding the major ?mbrial subunit FimA (type 1 ?mbriae) and FocA (F1C ?mbriae), and the two cryptic plasmids pMUT1 and pMUT2, and mean while the human E. coli Nissle 1917 was chosen as the positive control. The 427 bp-long plasmid fragment named pMUT2(a) was purified from the gel and made into DNA probe using digoxigenin random primer labeling. [Results] The PCR result showed that the expected fragments of the five specific primers target were amplified from the two of 135 DNA template samples and the potential presence of Nissle 1917 in pigs was initially confirmed. Using the method of in situ hybridization with prepared pMUT2(a) probe, two positive strains were screened from the two positive faecal samples. Two positive clones were finally confirmed as positive strains of Nissle 1917 through further experiments of serological test, PCR and sequencing. [Conclusion] The isolation and identification of swine-originated probiotics Nissle 1917 laid a foundation for further study and application of excellent animal originated probiotic.