Blake M R, Weimer B C
Department of Nutrition and Food Sciences, Utah State University, Logan 84322, USA.
Appl Environ Microbiol. 1997 May;63(5):1643-6. doi: 10.1128/aem.63.5.1643-1646.1997.
There are currently no methods for the rapid and sensitive detection of bacterial spores that could be used to direct raw materials containing high spore loads away from products that pose a food safety risk. Existing methods require an overnight incubation, cannot detect spores below 10(5) CFU/ml, or are not specific to particular species. This work describes a method to specifically detect < 10(4) CFU of bacterial spores per ml within 2 h. Polyclonal antibodies to Bacillus stearothermophilus spores were attached to 2.8-micron-diameter magnetic polystyrene beads by using a polythreonine cross-linker via the antibody carbohydrate moiety. A biotin-avidin-amplified sandwich enzyme-linked immunosorbent assay coupled to a fluorescent substrate was used to quantitate captured spores. The concentration of B. stearothermophilus spores in samples was linearly correlated to fluorescent activity (r2 = 0.99) with a lower detection limit of 8 x 10(3) CFU/ml and an upper detection limit of 8 x 10(5) CFU/ml. The detection limits are not fixed and can be changed by varying the immunomagnetic bead concentration. Several food and environmental samples were tested to demonstrate the versatility of the assay.
目前尚无能够快速、灵敏地检测细菌芽孢的方法，可用于避免将含有高芽孢载量的原材料用于存在食品安全风险的产品中。现有方法需要过夜培养，无法检测低于10⁵CFU/ml的芽孢，或者对特定物种不具有特异性。这项工作描述了一种在2小时内特异性检测每毫升低于10⁴CFU细菌芽孢的方法。通过使用聚苏氨酸交联剂经由抗体碳水化合物部分，将嗜热脂肪芽孢杆菌芽孢的多克隆抗体附着于直径2.8微米的磁性聚苯乙烯珠上。使用与荧光底物偶联的生物素-抗生物素蛋白放大夹心酶联免疫吸附测定法对捕获的芽孢进行定量。样品中嗜热脂肪芽孢杆菌芽孢的浓度与荧光活性呈线性相关（r² = 0.99），检测下限为8×10³CFU/ml，检测上限为8×10⁵CFU/ml。检测限不是固定的，可以通过改变免疫磁珠浓度来改变。测试了几种食品和环境样品以证明该测定法的通用性。
