MITOMYCIN IMMUNOBLOT COLONY ASSAY FOR DETECTION OF SHIGA-LIKE TOXIN-PRODUCING ESCHERICHIA-COLI IN FECAL SAMPLES - COMPARISON WITH DNA PROBES
MITOMYCIN IMMUNOBLOT COLONY ASSAY FOR DETECTION OF SHIGA-LIKE TOXIN-PRODUCING ESCHERICHIA-COLI IN FECAL SAMPLES - COMPARISON WITH DNA PROBES
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DOI：
10.1128/jcm.31.5.1167-1172.1993
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发表时间：
1993-05-01
影响因子：
9.4
通讯作者：
KEUSCH, GT
中科院分区：
文献类型：
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作者：
HULL, AE;ACHESON, DWK;KEUSCH, GT
我们开发了一种直接筛选免疫印迹测定法，用于检测粪便样本中产生志贺样毒素（SLT）的微生物。该测定利用了大肠杆菌中噬菌体介导的 SLT 产生特性以及丝裂霉素的噬菌体诱导作用。丝裂霉素的添加显着增加了可用于免疫检测的毒素量。通过使用丝裂霉素增强免疫印迹测定，可以将产生SLT的大肠杆菌与不产生毒素的大肠杆菌和正常粪便菌群以1:1,000至1:5,000的比例区分。免疫印迹测定在现场进行了检查，并与 SLT-I 和 SLT-II 的直接 DNA 探测进行了比较。该测定能够以高水平的灵敏度和特异性检测产生 SLT 的大肠杆菌。通过使用与 SLT-I 和 SLT-II B 亚基发生交叉反应的单克隆抗体代替多克隆抗毒素血清，特异性得到显着提高。我们的结论是，丝裂霉素增强免疫印迹集落测定是 DNA 探测的一种快速可靠的替代方法，用于检测粪便样本中噬菌体介导的 SLT 产生微生物，特别是当核酸探针的生产和使用不可行时。此外，它还可以分离阳性菌落以供进一步研究和确认。
We developed a direct screening immunoblot assay for the detection of Shiga-like toxin (SLT)-producing organisms in stool samples. The assay takes advantage of the phage-mediated nature of SLT production in Escherichia coli and the phage-inducing effects of mitomycin. The addition of mitomycin significantly enhanced the amount of toxin available for immunologic detection. By using the mitomycin-enhanced immunoblot assay, SLT-producing E. coli could be distinguished from non-toxin-producing E. coli and normal stool flora in ratios of 1:1,000 to 1:5,000. The immunoblot assay was examined in a field setting and compared with direct DNA probing for SLT-I and SLT-II.The assay was able to detect SLT-producing E. coli with a high level of sensitivity and specificity. Specificity was markedly improved by using a monoclonal antibody which cross-reacts with both SLT-I and SLT-II B subunits in place of the polyclonal antitoxin sera. We conclude that the mitomycin-enhanced immunoblot colony assay is a rapid and reliable alternative to DNA probing for the detection of phage-mediated SLT-producing organisms in stool samples, especially when the production and use of nucleic acid probes are not feasible. In addition, it permits isolation of positive colonies for further study and confirmation.