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目的探寻一种简单、快速、灵敏的检测艰难梭菌的方法。方法 以艰难梭菌毒素A和毒素B基因3’末端高度保守重复区分别设计一对引物,进行PCR反应,同时在同一体系相同反应条件下使用相同引物分别以大肠杆菌和乳杆菌DNA为模板进行扩增,比较二者结果。结果从艰难梭菌成功克隆了毒素A基因3’端重复区域960bp的基因片段及毒素B基因3’端重复区域1851bp的基因片段,不同来源的菌株出现了相同的2条电泳带,并通过测序鉴定;而对照的大肠杆菌和乳杆菌株无特异电泳带出现。结论从艰难梭菌毒素人毒素B基因3’末端重复区域克隆的基因片段具有保守性,可以作为基因探针在临床上进行艰难梭菌检测,该方法简便、灵敏,可直接用粪便标本进行检测。此外,这些基因编码的多肽具有较高的疏水性井存在着膜受体结合区域,可以此为基础进行基因工程蛋白质疫苗的研究。
Objective To explore a simple, rapid and sensitive method for detecting C. difficile. Methods A pair of primers was designed respectively for the highly conserved repeat regions of the 3 ’end of toxin A and toxin B genes of C. difficile to carry out PCR reaction. Meanwhile, the same primers were used under the same reaction conditions of the same system to take Escherichia coli and Lactobacillus DNA as template Amplify and compare the results. Results A 960bp gene fragment from the 3 ’repeat region of toxin A gene and 1851bp fragment from the 3’ repeat region of toxin B gene were successfully cloned from C. difficile. The same two bands appeared in different origins of the strain, Identification; while the control of E. coli and lactobacillus no specific electrophoresis bands appear. Conclusion The cloned gene fragment from the 3 ’end of Clostridium difficile toxin human toxin B gene is conservative and can be used as a gene probe to detect Clostridium difficile in clinics. The method is simple, sensitive and can be directly used for stool specimens detection . In addition, the polypeptides encoded by these genes have higher hydrophobicity and have membrane receptor binding domains. Based on this, studies on genetically engineered protein vaccines can be performed.