Wren B W, Clayton C L, Castledine N B, Tabaqchali S
Department of Medical Microbiology, St. Bartholomew's Hospital Medical College, West Smithfield, London, United Kingdom.
J Clin Microbiol. 1990 Aug;28(8):1808-12. doi: 10.1128/jcm.28.8.1808-1812.1990.
A 4.5-kilobase PstI fragment encoding part of the toxin A gene was isolated and used as a DNA probe in colony hybridization studies with 58 toxigenic and 17 nontoxigenic Clostridium difficile strains. All 58 toxigenic strains showed positive hybridization, in contrast to the 17 nontoxigenic strains. Southern blot analysis with the toxin A gene probe showed hybridization to a single fragment of equal intensities for HindIII-digested genomic DNAs isolated from C. difficile strains of wide-ranging toxin production. The positive hybridization signals were due to fragments of heterogeneous lengths (9 to 13 kilobases) for toxigenic strains of different types but were absent for the nontoxigenic strains. These results suggest the presence of a single copy of the toxin A gene on the genome of C. difficile strains, and the wide variation of toxin expression is not a reflection of gene copy number. The lack of toxin activity for nontoxigenic strains can be explained by the absence of at least part of the toxin A gene. The toxin A gene probe was tested against clostridial strains from 18 other species, of which only toxigenic C. sordellii strains showed positive hybridization. The specificity of the toxin A gene probe for toxigenic strains may lead to improved methods for the specific identification of toxigenic C. difficile strains from clinical specimens.
分离出一段编码毒素A基因部分序列的4.5千碱基对的PstI片段，并将其用作DNA探针，对58株产毒艰难梭菌菌株和17株无毒艰难梭菌菌株进行菌落杂交研究。与17株无毒菌株形成对比的是，所有58株产毒菌株均显示阳性杂交。用毒素A基因探针进行的Southern印迹分析表明，从毒素产生范围广泛的艰难梭菌菌株中分离出的经HindIII消化的基因组DNA与单个强度相等的片段发生杂交。阳性杂交信号是由于不同类型的产毒菌株的片段长度不同（9至13千碱基对），而无毒菌株则没有。这些结果表明，艰难梭菌菌株基因组上存在毒素A基因的单拷贝，毒素表达的广泛差异并非基因拷贝数的反映。无毒菌株缺乏毒素活性可以用至少部分毒素A基因的缺失来解释。用毒素A基因探针检测了来自其他18个物种的梭菌菌株，其中只有产毒的索氏梭菌菌株显示阳性杂交。毒素A基因探针对产毒菌株的特异性可能会导致改进从临床标本中特异性鉴定产毒艰难梭菌菌株的方法。
