Rice B E, Lamichhane C, Joseph S W, Rollins D M
Enteric Diseases Program, Naval Medical Research Institute, Bethesda, Maryland 20889, USA.
Clin Diagn Lab Immunol. 1996 Nov;3(6):669-77. doi: 10.1128/cdli.3.6.669-677.1996.
Contamination of retail poultry by Campylobacter spp. is a significant source of human diarrheal disease. We have developed a colony-lift immunoassay (CLI) for the detection of Campylobacter jejuni, C. coli, and C. lari isolated from such sources and grown on selective agar medium or on filter membranes. This technique has been successfully utilized to quantify Campylobacter colonies within 18 to 28 h after sampling. Hydrophobic, high-protein-binding membranes were prewet with methanol and used to imprint bacterial cells from the agar or filter membrane, while leaving colonies intact and viable. The membranes were air dried, peroxidase neutralized, blocked with bovine serum albumin in phosphate-buffered saline, and hybridized for 5 min with an affinity-purified, horseradish peroxidase-labeled goat anti-Campylobacter antibody preparation (Kirkegaard and Perry Laboratories). The membranes were washed briefly, exposed to a 3,'5,5'-tetramethylbenzidine membrane substrate, rinsed in deionized water, and allowed to dry. Lifted colonies of Campylobacter were identified by a blue color reaction on the membrane. Replicas of the membranes were made by marking the location of the Campylobacter colonies on clear transparencies, which were subsequently utilized to locate the original colony on the filter membrane or agar plate. The specificity of this antibody preparation has been evaluated against a wide range of Campylobacter spp., including American Type Culture Collection type and references strains, retail poultry isolates, and isolates obtained from cloacal swabs of live commercial broiler chickens. Specificity against numerous non-Campylobacter spp. obtained from the same sources was also evaluated. The CLI provided a rapid and simple means for detection and enumeration of enteropathogenic Campylobacter organisms. We have successfully combined this CLI procedure with methods recently developed in our laboratories for retail meat and poultry sampling. Potentially, broader applications for use of this technique include detection and enumeration of campylobacters from clinical, veterinary, and environmental samples.
弯曲杆菌属对零售家禽的污染是人类腹泻病的一个重要来源。我们开发了一种集落印迹免疫测定法（CLI），用于检测从这类来源分离并在选择性琼脂培养基或滤膜上生长的空肠弯曲杆菌、大肠弯曲杆菌和拉氏弯曲杆菌。该技术已成功用于在采样后18至28小时内对弯曲杆菌菌落进行定量。疏水的、高蛋白结合膜先用甲醇预湿，用于从琼脂或滤膜上印迹细菌细胞，同时使菌落保持完整且有活力。膜在空气中干燥，用过氧化物酶中和，用磷酸盐缓冲盐水中的牛血清白蛋白封闭，并用亲和纯化的辣根过氧化物酶标记的山羊抗弯曲杆菌抗体制剂（Kirkegaard和Perry实验室）杂交5分钟。膜短暂洗涤，暴露于3',5,5'-四甲基联苯胺膜底物，用去离子水冲洗，然后干燥。弯曲杆菌的印迹菌落通过膜上的蓝色反应来鉴定。通过在透明胶片上标记弯曲杆菌菌落的位置制作膜的复制品，随后利用这些复制品在滤膜或琼脂平板上定位原始菌落。已针对多种弯曲杆菌属对该抗体制剂的特异性进行了评估，包括美国典型培养物保藏中心的类型和参考菌株、零售家禽分离株以及从活的商业肉鸡泄殖腔拭子中获得的分离株。还评估了针对从相同来源获得的众多非弯曲杆菌属的特异性。CLI为检测和计数肠道致病性弯曲杆菌提供了一种快速简便的方法。我们已成功将此CLI程序与我们实验室最近开发的零售肉类和家禽采样方法相结合。该技术潜在的更广泛应用包括检测和计数临床、兽医和环境样本中的弯曲杆菌。
