Gicquelais K G, Baldini M M, Martinez J, Maggi L, Martin W C, Prado V, Kaper J B, Levine M M
Center for Vaccine Development, University of Maryland School of Medicine, Baltimore 21201.
J Clin Microbiol. 1990 Nov;28(11):2485-90. doi: 10.1128/jcm.28.11.2485-2490.1990.
A simple and economical method was developed for using biotinylated DNA probes to hybridize with bacterial colonies belonging to the various categories of diarrhea-causing Escherichia coli. Simplification and cost containment were achieved by using Whatman no. 541 filter papers instead of nitrocellulose, by minimizing the concentration of proteinase K (an expensive but necessary reagent used to pretreat the colony blots prior to hybridization with biotin-labeled DNA probes) and by reusing hybridization solution containing labeled probe DNA. After exposing the colony blots to lysing solution and steam, followed by lysozyme (1.5 mg/ml), sucrose (25%), and proteinase K (10 micrograms/ml) treatments, biotinylated probes were used to detect enterotoxigenic, enteropathogenic, enterohemorrhagic, diffuse adherence, and enteroinvasive categories of diarrhea-causing E. coli with a high level of sensitivity and specificity. Three independent observers who were experienced in reading DNA blots recorded remarkably similar results, while less satisfactory results were obtained when the blots were read by an inexperienced observer. This technique will be useful in laboratories in which radioactive isotopes are unavailable or impractical and in which budgets are restricted.
开发了一种简单且经济的方法，用于使用生物素化的DNA探针与属于各类致泻性大肠杆菌的细菌菌落进行杂交。通过使用Whatman 541滤纸代替硝酸纤维素、将蛋白酶K（一种用于在与生物素标记的DNA探针杂交前预处理菌落印迹的昂贵但必要的试剂）的浓度降至最低以及重复使用含有标记探针DNA的杂交溶液，实现了简化和成本控制。在将菌落印迹暴露于裂解液和蒸汽中，然后进行溶菌酶（1.5 mg/ml）、蔗糖（25%）和蛋白酶K（10微克/ml）处理后，使用生物素化探针检测致泻性大肠杆菌的产肠毒素型、肠致病性、肠出血性、弥漫性黏附型和肠侵袭型，具有高度的敏感性和特异性。三位有解读DNA印迹经验的独立观察者记录的结果非常相似，而由无经验的观察者解读印迹时得到的结果则不太令人满意。该技术将在无法获得或不适用放射性同位素且预算有限的实验室中有用。
