Thomas A, Smith H R, Willshaw G A, Rowe B
Division of Enteric Pathogens, Central Public Health Laboratory, London, England.
Mol Cell Probes. 1991 Apr;5(2):129-35. doi: 10.1016/0890-8508(91)90007-7.
Vero cytotoxin producing Escherichia coli (VTEC) were detected in faecal specimens and bacterial isolates, using non-radioactively labelled polynucleotide and oligonucleotide DNA probes specific for Vero cytotoxin (VT) genes. VT1 and VT2 structural gene sequences, previously cloned and used for radioactive probes, were labelled with digoxigenin or biotin. Oligonucleotide gene sequences coding for the A subunit of VT1, VT2 and VT2 variant were labelled with digoxigenin. The VT1 and VT2 probes were specific for detecting VT1 and VT2 gene sequences and gave very similar results to those obtained using the radioactive label 35S as a standard. The VT2 variant probe hybridized only with the strains of porcine origin. For the range of isolates tested, there was little significant difference in specificity and sensitivity between the digoxigenin-labelled polynucleotide and oligonucleotide probes. The biotin system gave rise to more non-specific effects, particularly with some non-E. coli strains, and was therefore less reliable. All of the digoxigenin-labelled probes gave satisfactory results after several times re-use, which is of importance when considering cost.
使用对Vero细胞毒素（VT）基因特异的非放射性标记的多核苷酸和寡核苷酸DNA探针，在粪便标本和细菌分离物中检测到产Vero细胞毒素的大肠杆菌（VTEC）。先前克隆并用于放射性探针的VT1和VT2结构基因序列，用洋地黄毒苷或生物素进行标记。编码VT1、VT2和VT2变体A亚基的寡核苷酸基因序列用洋地黄毒苷进行标记。VT1和VT2探针特异于检测VT1和VT2基因序列，并且与以放射性标记35S作为标准所获得的结果非常相似。VT2变体探针仅与猪源菌株杂交。对于所测试的分离物范围，洋地黄毒苷标记的多核苷酸和寡核苷酸探针之间在特异性和敏感性上几乎没有显著差异。生物素系统产生更多非特异性效应，尤其是与一些非大肠杆菌菌株，因此可靠性较差。所有洋地黄毒苷标记的探针在多次重复使用后都给出了满意的结果，这在考虑成本时很重要。
