Piérard D, Muyldermans G, Moriau L, Stevens D, Lauwers S
Department of Microbiology, VTEC Reference Laboratory, Academisch Ziekenhuis Vrije Universiteit Brussel, Brussels, Belgium.
J Clin Microbiol. 1998 Nov;36(11):3317-22. doi: 10.1128/JCM.36.11.3317-3322.1998.
The sequence of a verocytotoxin 2 (VT2) variant gene that was untypeable by the B subunit PCR and restriction fragment length polymorphism analysis (PCR-RFLP) method described by Tyler et al. (S. D. Tyler, W. M. Johnson, H. Lior, G. Wang, and K. R. Rozee, J. Clin. Microbiol. 29:1339-1343, 1991) was determined and compared with published sequences. It was highly homologous to two recently reported VT2 variant sequences. The PCR-RFLP method described by Tyler et al. was extended to include these new sequences. New VT2 variants were identified in 65 of 359 VT-producing Escherichia coli (VTEC) with newly designed primers (VT2-cm and VT2-f) and were characterized as well by restriction analysis of the amplification products obtained with another VT2-specific primer pair (VT2-e and VT2-f). The VT genes harbored by 64 of these isolates proved to be untypeable by Tyler's PCR-RFLP method because no amplification was obtained with the primers used with this method (VT2-c and VT2-d). The last isolate harbored the new variant gene in addition to VT2vh-a. None of the isolates harboring these new toxin genes belonged to serogroups O157, O26, O103, O111, and O145. All 65 isolates were negative for the eaeA gene and were significantly less frequently enterohemolytic or positive for the enterohemorrhagic E. coli (EHEC) virulence plasmid than non-O157 VTEC isolates harboring other VT2 genes. They were also less frequently isolated from patients with EHEC-associated symptoms. The extended PCR-RFLP typing method is a useful tool to identify less-virulent VTEC isolates and for VT genotyping in epidemiological studies with non-O157 strains.
对一种用泰勒等人（S. D. 泰勒、W. M. 约翰逊、H. L. 利奥、G. 王和K. R. 罗泽，《临床微生物学杂志》29:1339 - 1343，1991年）描述的B亚基聚合酶链反应（PCR）和限制性片段长度多态性分析（PCR - RFLP）方法无法分型的志贺毒素2（VT2）变体基因的序列进行了测定，并与已发表的序列进行了比较。它与最近报道的两个VT2变体序列高度同源。泰勒等人描述的PCR - RFLP方法得到扩展，纳入了这些新序列。用新设计的引物（VT2 - cm和VT2 - f）在359株产VT的大肠杆菌（VTEC）中的65株中鉴定出了新的VT2变体，并通过用另一对VT2特异性引物（VT2 - e和VT2 - f）对扩增产物进行限制性分析进行了表征。这些分离株中的64株所携带的VT基因用泰勒的PCR - RFLP方法无法分型，因为用该方法使用的引物（VT2 - c和VT2 - d）未获得扩增产物。最后一株分离株除了携带VT2vh - a外，还携带新的变体基因。携带这些新毒素基因的分离株均不属于血清型O157、O26、O103、O111和O145。所有65株分离株的eaeA基因均为阴性，与携带其他VT2基因的非O157 VTEC分离株相比，肠溶血或肠出血性大肠杆菌（EHEC）毒力质粒呈阳性的频率显著更低。它们从有EHEC相关症状的患者中分离到的频率也较低。扩展的PCR - RFLP分型方法是鉴定毒性较低的VTEC分离株以及在非O157菌株的流行病学研究中进行VT基因分型的有用工具。
