新疆牛源大肠杆菌O157∶H7的分离鉴定及ERIC-PCR基因分型研究

doi:10.16431/j.cnki.1671-7236.2020.01.022

摘要/Abstract

摘要： 为了了解牛源大肠杆菌（E.coli）O157∶H7在新疆地区的污染状况以及遗传多样性，探究不同地区分离菌株的遗传关系，为控制牛源E.coli O157∶H7的传播提供试验依据。将采集的样品在EC肉汤中进行增菌（37 ℃、180 r/min），接着将增菌液划线接种到SMAC平板上，37 ℃培养箱中过夜培养18 h左右。挑取SMAC平板上白色或无色单菌落接种MUG培养基，37 ℃培养18 h左右，将无荧光样品接种到SMAC平板上，37 ℃培养18 h左右，隔天挑取白色或无色单菌落进行PCR鉴定，具有rfbE和fliC基因条带的即为阳性菌株。将阳性菌株进行肠杆菌基因间重复共有序列扩增（ERIC-PCR）指纹图谱聚类分析，分析菌株之间的同源性关系。ERIC-PCR结果显示，相似性100%的菌株有3组。从伊犁地区分离到的菌株差异性最大，具有6种分型；其次是乌鲁木齐，具有4种分型。菌株来源多样性最多的在D簇，由此可见通过ERIC-PCR分型，可以进行溯源观察。ERIC-PCR能够区分特定采样点或物种的分离物，它能够证明从不同来源的菌株之间，存在着某些相似的ERIC特性，并聚集在同一个簇群中。该研究中筛选出的E.coli O157∶H7菌株具有广泛的遗传多样性，该方法对于检测不同物种间的细菌差异非常敏感。由此可见ERIC-PCR可以作为E.coli O157∶H7常规监测和鉴定的一个有效的工具。

关键词: 牛; E.coli O157∶H7; ERIC-PCR; 遗传关系; 基因分型

Abstract: In order to understand the pollution status and genetic diversity of bovine Escherichia coli O157:H7 in Xinjiang,to explore the genetic relationship of isolates from different areas,and to provide experimental basis for controlling the transmission of bovine E.coli O157:H7 in cattle,in this experiment,the samples were added to EC broth (37 ℃,180 r/min),then inoculated on SMAC plate and cultured overnight in 37 ℃ incubator for about 18 h.Then,white or colorless single colonies on SMAC were inoculated the MUG medium,and cultured at 37 ℃ for 18 h.The non-fluorescent samples were inoculated on the SMAC plate,and cultured at 37 ℃ for about 18 h.The white or colorless single colonies were selected the next day for PCR identification.The positive strains with rfbE and fliC gene bands were identified.ERIC-PCR fingerprinting was used to analyze the homology among the positive strains.ERIC-PCR results showed that there were 3 groups of strains with 100% similarity.The strains isolated from the Yili region were the most diverse,with 6 types,followed by Urumqi with 4 types.The most diverse source of strains was in cluster D.It could be traced by ERIC-PCR typing.ERIC-PCR was able to distinguish the isolates of specific sampling points or species.It could demonstrate that there were some similar ERIC characteristics among strains from different sources and they were clustered in the same cluster.The E.coli O157:H7 strain selected in this study had extensive genetic diversity,which was very sensitive to detect bacterial differences among different species.Therefore,ERIC-PCR could be used as an effective tool for routine monitoring and identification of E.coli O157:H7.

Key words: cattle; E.coli O157:H7; ERIC-PCR; genetic relationship; genotyping

中图分类号:

S852.61

苏战强, 张凌, 刘璐瑶, 王栋, 马晓玉, 艾柯代·吐鲁洪, 张毅, 佟盼盼. 新疆牛源大肠杆菌O157∶H7的分离鉴定及ERIC-PCR基因分型研究[J]. 中国畜牧兽医, 2020, 47(1): 182-189.

SU Zhanqiang, ZHANG Ling, LIU Luyao, WANG Dong, MA Xiaoyu, AIKEDAI·TULUHONG, ZHANG Yi, TONG Panpan. Isolation,Identification and ERIC-PCR Genotyping of E.coli O157:H7 from Xinjiang Cattle[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(1): 182-189.

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