鉴别布鲁氏菌S2疫苗株斑点杂交检测方法的建立

发布时间：2018-03-04 01:11

  本文选题：布鲁氏菌病　切入点：布鲁氏菌　出处：《内蒙古农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：为了鉴别布鲁氏菌S2疫苗株与其它布鲁氏菌,本研究建立了鉴别布鲁氏菌S2疫苗株的斑点杂交法。针对布鲁氏菌S2疫苗株IclR基因序列保守区与其他菌株的差异,设计合成针对S2疫苗株的大小为29 bp的寡核苷酸探针,并进行地高辛标记。根据探针作用位点设计引物,对扩增的PCR产物经过回收、纯化、定量,变性后经过120℃30 min烘烤固定在NC膜上,然后与探针杂交、显色。结果显示,本实验设计的PCR引物1能够对S2疫苗株扩增出约330 bp的核酸片段,对其他参考布鲁氏菌扩增出约360 bp的核酸片段；采用末端标记法可以成功地将地高辛连接到本研究设计合成的寡核苷酸探针上,标记效果良好；探针只能和S2的PCR产物杂交,特异性强,重复性好,最低能够检测到10 pg的PCR片段,能够在10h内鉴别出布鲁氏菌S2疫苗株。
[Abstract]:In order to identify Brucella S2 vaccine strain from other brucella strains, a dot hybridization method was established to identify the Brucella S2 vaccine strain. The difference of IclR gene conserved region between Brucella S2 vaccine strain and other strains was studied. A 29bp oligonucleotide probe was designed and synthesized for S2 vaccine strain and labeled with digoxigenin. Primers were designed according to the action site of the probe to recover, purify and quantify the amplified PCR products. After denaturation, the NC membrane was roasted at 120 鈩，

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