Tortorello M L, Stewart D S, Cray W C
National Center for Food Safety and Technology, U.S. Food and Drug Administration, Summit-Argo, IL 60501, USA.
Vet Microbiol. 1996 Aug;51(3-4):343-9. doi: 10.1016/0378-1135(96)00041-7.
The antibody-direct epifluorescent filter technique (Ab-DEFT) was adapted for direct detection of Escherichia coli O157:H7 in bovine feces. The method involved suspension of bovine feces in buffer, centrifugation for 30 s, treatment of the supernatant with trypsin and Triton X-100 at 50 degrees C for 10 min, pre-filtration through 5 and 1.2 microns pore filters, and filtration through a 0.4 micron pore filter. The final filter was stained with fluorescein-labeled polyclonal antibody specific for the O157 antigen and examined by epifluorescence microscopy. The Ab-DEFT was correlated with viable plate counts for enumeration of the pathogen in artificially inoculated bovine feces (r = 0.96). The limit of detection was approximately 10(4)-10(5) CFU/g feces. The procedure provided a clean background for microscopic visualization of cells; however, cell loss and inaccurate quantitation sometimes resulted. E. coli O157:H7 was detected in feces of an inoculated calf for more than 3 weeks post-inoculation. The Ab-DEFT may be useful for rapid screening of cattle for the presence of the E. coli O157:H7 and as an analytical method in ecological studies of the pathogen.
抗体直接荧光过滤技术（Ab-DEFT）被用于直接检测牛粪便中的大肠杆菌O157:H7。该方法包括将牛粪便悬浮于缓冲液中，离心30秒，将上清液在50摄氏度下用胰蛋白酶和 Triton X-100处理10分钟，先通过5微米和1.2微米孔径的滤膜预过滤，再通过0.4微米孔径的滤膜过滤。最后一张滤膜用针对O157抗原的荧光素标记多克隆抗体染色，并用落射荧光显微镜检查。在人工接种牛粪便中，Ab-DEFT与活菌平板计数法用于病原体计数具有相关性（r = 0.96）。检测限约为每克粪便10(4)-10(5) CFU。该程序为细胞的显微镜观察提供了清晰的背景；然而，有时会导致细胞损失和定量不准确。在接种后的小牛粪便中，接种后超过3周都检测到了大肠杆菌O157:H7。Ab-DEFT可用于快速筛查牛群中是否存在大肠杆菌O157:H7，也可作为该病原体生态研究中的一种分析方法。
