Ackermann G, Tang Y J, Henderson J P, Rodloff A C, Silva J, Cohen S H
Department of Internal Medicine, Division of Infectious and Immunologic Diseases, University of California-Davis, PSSB Suite 500, Sacramento, CA 95817, USA.
Mol Cell Probes. 2001 Oct;15(5):301-6. doi: 10.1006/mcpr.2001.0373.
Toxigenic Clostridium difficile is the etiologic agent of C. difficile-associated diarrhoea (CDAD), the most common cause of hospital-acquired infectious diarrhoea. The genes tcdA and tcdB, which encode for the toxin A and B proteins, are part of the pathogenicity locus (PaLoc) of toxigenic C. difficile. Genetic and virulence studies at the molecular level in C. difficile have been hindered by the lack of techniques for DNA manipulation in this species. We describe the electroporation of DNA fragments from a toxigenic isolate into a non-toxigenic strain of C. difficile. Using previously described methods of electroporation into Clostridium spp., the complete toxin B gene and polymerase chain reaction (PCR) fragments of the PaLoc were cloned and electroporated into a non-toxigenic strain of C. difficile. The resulting transformed clones were screened for the introduced gene fragments by PCR, which confirmed their presence. This is the first description of introduction of DNA into C. difficile by electroporation.
产毒艰难梭菌是艰难梭菌相关性腹泻（CDAD）的病原体，是医院获得性感染性腹泻最常见的病因。编码毒素A和B蛋白的tcdA和tcdB基因是产毒艰难梭菌致病位点（PaLoc）的一部分。由于缺乏对该菌种进行DNA操作的技术，艰难梭菌分子水平的遗传和毒力研究受到了阻碍。我们描述了将来自产毒分离株的DNA片段电穿孔导入艰难梭菌无毒菌株的过程。使用先前描述的电穿孔导入梭菌属的方法，克隆了完整的毒素B基因和PaLoc的聚合酶链反应（PCR）片段，并将其电穿孔导入艰难梭菌无毒菌株。通过PCR对所得转化克隆进行引入基因片段的筛选，证实了它们的存在。这是首次描述通过电穿孔将DNA导入艰难梭菌的过程。
